Pullulanase variants and methods for preparing such variants with predetermined properties

ABSTRACT

The present invention relates to a method for producing a variant of a parent pullulanase, the variant having at least one altered property as compared to the parent pullulanase. The invention also relates to pullulanase variants and to the use of pullulanase variants of the invention for use in particular starch conversion processes.

FIELD OF THE INVENTION

[0001] The present invention relates to variants of pullulanases and tomethods for constructing such variants with altered properties,including stability (e.g., thermostability), pH dependent activity,substrate specificity, such as increased isoamylase activity, orspecific activity; specific activity at a given pH and/or alteredsubstrate specificity, such as an altered pattern of substrate cleavageor an altered pattern of substrate inhibition.

BACKGROUND OF THE INVENTION

[0002] Starches such as corn, potato, wheat, manioc and rice starch areused as the starting material in commercial large-scale production ofsugars, such as high fructose syrup, high maltose syrup, maltodextrins,amylose, G4-G6 oligosaccharides and other carbohydrate products such asfat replacers.

[0003] Degradation of Starch Starch usually consists of about 80%amylopectin and 20% amylose. Amylopectin is a branched polysaccharide inwhich linear chains alpha-1,4 D-glucose residues are joined by alpha-1,6glucosidic linkages. Amylopectin is partially degraded by alpha-amylase,which hydrolyzes the 1,4-alpha-glucosidic linkages to produce branchedand linear oligosaccharides. Prolonged degradation of amylopectin byalpha-amylase results in the formation of so-called alpha-limit dextrinsthat are not susceptible to further hydrolysis by the alpha-amylase.Branched oligosaccharides can be hydrolyzed into linear oligosaccharidesby a debranching enzyme. The remaining branched oligosaccharides can bedepolymerized to D-glucose by glucoamylase, which hydrolyzes linearoligosaccharides into D-glucose.

[0004] Amylose is a linear polysaccharide built up of D-glucopyranoseunits linked together by alpha-1,4 glucosidic linkages. Amylose isdegraded into shorter linear oligosaccharides by alpha-amylase, thelinear oligosaccharides being depolymerized into D-glucose byglucoamylase.

[0005] In the case of converting starch into a sugar, the starch isdepolymerized. The depolymerization process consists of a pretreatmentstep and two or three consecutive process steps, namely a liquefactionprocess, a saccharification process and, depending on the desired endproduct, optionally an isomerization process.

[0006] Pre-Treatment of Native Starch

[0007] Native starch consists of microscopic granules that are insolublein water at room temperature. When an aqueous starch slurry is heated,the granules swell and eventually burst, dispersing the starch moleculesinto the solution. During this “gelatinization” process there is adramatic increase in viscosity. As the solids level is 30-40% in atypical industrial process, the starch has to be thinned or “liquefied”so that it can be handled. This reduction in viscosity is today mostlyobtained by enzymatic degradation.

[0008] Liquefaction

[0009] During the liquefaction step, the long-chained starch is degradedinto smaller branched and linear units (maltodextrins) by analpha-amylase (e.g., Termamyl™, available from Novozymes A/S, Denmark).The liquefaction process is typically carried out at about 105-110° C.for about 5 to 10 minutes followed by about 1-2 hours at about 95° C.The pH generally lies between about 5.5 and 6.2. In order to ensureoptimal enzyme stability under these conditions, calcium is added, e.g.,1 mM of calcium (40 ppm free calcium ions). After this treatment theliquefied starch will have a “dextrose equivalent” (DE) of 10-15.

[0010] Saccharification

[0011] After the liquefaction process the maltodextrins are convertedinto dextrose by addition of a glucoamylase (e.g. AMG™, available fromNovozymes A/S) and a debranching enzyme, such as an isoamylase (see,e.g., U.S. Pat. No. 4,335,208) or a pullulanase (e.g., Promozyme®,available from Novozymes A/S; see U.S. Pat. No. 4,560,651). Before thisstep the pH is reduced to a value below 4.5, e.g. about 3.8, maintainingthe high temperature (above 95° C.) for a period of, e.g., about 30 min.to inactivate the liquefying alpha-amylase to reduce the formation ofshort oligosaccharides called “panose precursors” which cannot behydrolyzed properly by the debranching enzyme.

[0012] The temperature is then lowered to 60° C., glucoamylase anddebranching enzyme are added, and the saccharification process proceedsfor about 24-72 hours.

[0013] Normally, when denaturing the alpha-amylase after theliquefaction step, a small amount of the product comprises panoseprecursors, which cannot be degraded by pullulanases or AMG. If activeamylase from the liquefaction step is present during saccharification(i.e., no denaturing), this level can be as high as 1-2% or even higher,which is highly undesirable as it lowers the saccharification yieldsignificantly. For this reason, it is also preferred that thealpha-amylase is one which is capable of degrading the starch moleculesinto long, branched oligosaccharides (such as, e.g., the Fungamyl™-likealpha-amylases) rather than shorter branched oligosaccharides.

[0014] Isomerization

[0015] When the desired final sugar product is, e.g., high fructosesyrup, the dextrose syrup may be converted into fructose by enzymaticisomerization. After the saccharification process the pH is increased toa value in the range of 6-8, preferably about pH 7.5, and the calcium isremoved by ion exchange. The dextrose syrup is then converted into highfructose syrup using, e.g., an immobilized glucose isomerase (such asSweetzyme™, available from Novozymes A/S).

[0016] Debranching Enzymes

[0017] Debranching enzymes which can attack amylopectin are divided intotwo classes: isoamylases (E C. 3.2.1.68) and pullulanases (E C.3.2.1.41), respectively. Isoamylase hydrolyses alpha-1,6-D-glucosidicbranch linkages in amylopectin and beta-limit dextrins and can bedistinguished from pullulanases by the inability of isoamylase to attackpullulan, and by their limited action on alpha-limit dextrins.

[0018] When an acidic stabilized alpha-amylase is used for the purposeof maintaining the amylase activity during the entire saccharificationprocess (no inactivation), the degradation specificity should be takeninto consideration. It is desirable in this regard to maintain thealpha-amylase activity throughout the saccharification process, sincethis allows a reduction in the amyloglucidase addition, which iseconomically beneficial and reduces the AMG™ condensation productisomaltose, thereby increasing the DE (dextrose equivalent) yield.

[0019] It will be apparent from the above discussion that the knownstarch conversion processes are performed in a series of steps, due tothe different requirements of the various enzymes in terms of, e.g.,temperature and pH. It would therefore be desirable to be able toengineer one or more of these enzymes, e.g., pullulanases, so that theoverall process could be performed in a more economical and efficientmanner. One possibility in this regard is to engineer the otherwisethermolabile pullulanases so as to render them more stable at highertemperatures.

BRIEF DISCLOSURE OF THE INVENTION

[0020] The inventors have modified the amino acid sequence of apullulanase to obtain variants with improved properties, based on thethree-dimensional structure of the pullulanase Promozyme® (availablefrom Novozymes A/S, Denmark). The variants have altered physicochemicalproperties, e.g., an altered pH optimum, improved thermostability,increased specific activity or an altered cleavage pattern.

[0021] Accordingly, the object of the present invention is to provide amethod for constructing pullulanases having altered properties, inparticular to provide a method for constructing pullulanases havingimproved thermostability, altered pH dependent activity and/or alteredsubstrate specificity, such as increased isoamylase activity.

[0022] Thus, in its broadest aspect, the present invention relates to amethod for constructing a variant of a parent pullulanase, wherein thevariant has at least one altered property as compared to said parentpullulanase, which method comprises:

[0023] i) analyzing the structure of the pullulanase to identify, on thebasis of an evaluation of structural considerations, at least one aminoacid residue or at least one structural region of the pullulanase, whichis of relevance for altering said property;

[0024] ii) constructing a variant of the pullulanase, which as comparedto the parent pullulanase, has been modified in the amino acid residueor structural part identified in i) so as to alter said property; and

[0025] iii) testing the resulting pullulanase variant for said property.

[0026] The property that may be altered by the above methods of thepresent invention may be, e.g., thermostability, pH dependent activity,specific activity, or substrate specificity. Thus, the variant may have,e.g., increased thermostability, higher activity at a lower pH, analtered pH optimum, improved thermostability, or increased specificactivity, such as increased isoamylase activity.

[0027] Although it has been described in the following that modificationof the parent pullulanase in certain regions and/or positions isexpected to confer a particular effect to the thus produced pullulanasevariant (such as an improved thermostability or an increased isoamylaseactivity), it should be noted that modification of the parentpullulanase in any of such regions may also give rise to any other ofthe above-mentioned effects. For example, any of the regions and/orpositions mentioned as being of particular interest with respect to,e.g., improved thermostability, may also give rise to, e.g., higheractivity at a lower pH, an altered pH optimum, or increased specificactivity, such as increased isoamylase activity.

[0028] Further aspects of the present invention relates to variants of apullulanase, the DNA encoding such variants and methods of preparing thevariants. Still further aspects of the present invention relates to theuse of the variants for various industrial purposes, in particular forprocesses where sweeteners are made from starch. Other aspects of thepresent invention will be apparent from the below description as well asfrom the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 shows the DNA plasmid pCA36 harboring the gene encoding thepullulanase from Bacillus deramificans (SEQ ID NO: 0.3).

[0030] APPENDIX 1 shows the structural coordinates for the solvedcrystal structure of Promozyme®

[0031] APPENDIX 2 shows a sequence alignment between Promozyme® (SEQ IDNO: 2), the pullulanase from Bacillus deramificans (SEQ ID NO: 4), andthe pullulanase from Bacillus acidopullulyticus (SEQ ID NO: 6) describedin FEMS Mic. Let. (1994) 115, 97-106.

DETAILED DISCLOSURE OF THE INVENTION

[0032] Pullulanases

[0033] As explained above, pullulanases are enzymes classified in EC3.2.1.41 and such enzymes are characterized by their ability tohydrolyze the alpha-1,6-glycosidic bonds in, for example, amylopectinand pullulan.

[0034] A particularly interesting pullulanase is the pullulanase fromBacillus acidopullulyticus described in U.S. Pat. No. 4,560,651(hereinafter referred to as Promozyme®). Promozyme® has the amino acidsequence set forth in amino acids 1-921 of SEQ ID NO: 1. Thethree-dimensional structure of Promozyme® is described below.

[0035] Another interesting pullulanase is the pullulanase from Bacillusderamificans described in U.S. Pat. No. 5,736,375. This enzyme has theamino acid sequence set forth in amino acid sequence 1-928 of SEQ ID NO:3. Homology building of the tree-dimensional structure of theabove-mentioned pullulanase is described below.

[0036] In general, a preferred pullulanase suitable for the purposedescribed herein should have one or more of the following properties:

[0037] i) A three-dimensional structure homologous to Promozyme®.

[0038] ii) An amino acid sequence which is at least 40% homologous toSEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 50%, e.g., at least60%, such as a least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%homologous to SEQ ID NO:1 or SEQ ID NO: 3.

[0039] iii) A nucleic acid sequence which hybridizes to the nucleic acidsequence set forth in SEQ ID NO:1 or SEQ ID NO:3.

[0040] The structural homology referred to above in i) above is based onother sequence homologies, hydrophobic cluster analysis or by reversethreading (Huber, T; Torda, A E, PROTEIN SCIENCE Vol. 7, No. 1 pp.142-149 (1998)) and which by any of these methods is predicted to havethe same tertiary structure as Promozyme®, wherein the tertiarystructure refers to the overall folding or the folding of Domains N1,N2, A, B, and C. Alternatively, a structural alignment betweenPromozyme®and homologous sequences may be used to identify equivalentpositions.

[0041] For example, the homology between various pullulanase with knownamino acid sequence has been compiled in the below matrix: 1 2 3 4 5 6 78 9 10  1. pula_kleae 100 86 59 51 52 53 52 52 55 50  2. pula_klepn 10058 51 51 53 53 53 53 52  3. w81973 100 55 56 52 55 54 51 56  4. r56989100 98 60 76 54 56 76  5. sp929mat 100 61 78 54 57 78  6. fervido_x 10061 57 54 62  7. sp734 100 56 54 91  8. r71616 100 54 56  9. w09257 10054 10. Promozyme ® 100

[0042] The above homology calculations were determined by use of the GAPprogram from the UWGCG package using default values for GAP penalties,i.e., GAP creation penalty of 3.0 and GAP extension penalty of 0.1(Program Manual for the Wisconsin Package, Version 8, August 1994,Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711).

[0043] A sequence alignment between Promozyme® (SEQ ID NO: 1), thepullulanase from Bacillus deramificans (SEQ ID NO: 3) and thepullulanase from Bacillus acidopullulyticus (SEQ ID NO: 5) described inFEMS Mic. Let. (1994) 115, 97-106, is shown in Appendix 2.

[0044] Three-Dimensional Structure of Pullulanase

[0045] Promozyme® was used to elucidate the three-dimensional structureforming the basis for the present invention.

[0046] The structure of Promozyme® was solved in accordance with theprinciple for x-ray crystallographic methods, for example, as given inX-Ray Structure Determination, Stout, G. K. and Jensen, L. H., JohnWiley & Sons, Inc. NY, 1989.

[0047] The structural coordinates for the solved crystal structure ofPromozyme® using the isomorphous replacement method are given instandard PDB format (Protein Data Bank, Brookhaven National Laboratory,Brookhaven, Conn.) as set forth in Appendix 1. It is to be understoodthat Appendix 1 forms part of the present application. In the context ofAppendix 1, the following abbreviations are used: WAT refers to water orto calcium. Amino acid residues are given in their standard three-lettercode.

[0048] The structure of said Pullulanase is made up of five globulardomains, ordered N1 being 1-310 (which may be subdivided into N1′ beingresidues 1-111, N1″ being residues 112-158 and 261-310, and N1′″ beingresidues 159-261), N2, A, B, and C. The domains can be defined as beingresidues 1-310 for domain N1, 311-420 for Domain N2, residues 421-556and 596-835 for domain A, residues 557-595 for Domain B, residues596-922 for Domain C, wherein the numbering refers to the amino acidsequence in SEQ ID NO: 1. Features of Domains N1, A, B and C ofparticular interest are described below.

[0049] Domain N1

[0050] Domain N1 contains in this particular pullulanase an extra loopof 100 residues compared to the pullulanase from Bacillusacidopullulyticus having the amino acid sequence shown in SEQ ID NO: 5.The loop is also present in the pullulanase from Bacillus deramificanshaving the amino acid sequence shown in SEQ ID NO: 3.

[0051] Part of the N2 domain is homologeous to the N1 domain ofPseudomonase amyloderamosa isoamylase (1bf2pdb from Brookhavendatabase).

[0052] Domain A

[0053] Domain A is the largest domain and contains the active site whichcomprises a cluster of three amino acid residues, D622, D736 and E651,spatially arranged at the bottom of a cleft in the surface of theenzyme. The structure of Domain A shows an overall fold in common withthe alpha-amylases for which the structure is known, viz. the(beta/alpha) 8 barrel with eight central beta strands (numbered 1-8) andeight flanking alpha-helices. The beta-barrel is defined by McGregor, J.Prot. Chem. 7:399, 1988. The C-terminal end of the beta strand 1 isconnected to helix 1 by a loop denoted loop 1 and an identical patternis found for the other loops, although the loops show some variation insize and some can be quite extensive.

[0054] The eight central beta-strands in the (beta/alpha) 8 barrelsuperimpose reasonably well with the known structures of family 13(Henrissat B. Biochem. J. (1991) 280, 309-316 and Henrissat B. andBairoch A. Biochem. J. (1993) 293, 781-788). This part of the structure,including the close surroundings of the active site located at theC-terminal end of the beta-strands, shows a high degree of homology withisoamylases.

[0055] In contrast, the loops connecting the beta-strands and alphahelices display a high degree of variation from the known structures offamily 13 enzymes. These loops constitute the structural context of theactive site, and the majority of the contacts to the substrate are foundamong residues located in these loops. Distinguishing characteristicssuch as substrate specificity, substrate binding, pH activity profile,substrate cleavage pattern, and the like, are determined by specificamino acids and the positions they occupy in these loops.

[0056] Domain B

[0057] Domain B, also referred to as loop 3 of the (beta/alpha) 8barrel, in comprises amino acid residues 557-595 of the amino acidsequence shown in SEQ ID NO: 1. The most striking difference to otherfamily 13 enzymes is the short amino acid sequence. This short sequenceloop are of the same size as the isoamylase loop 3 and spatiallypositioned close to the active site residues and in close contact to thesubstrate.

[0058] Domain C

[0059] Domain C in Promozyme® comprises amino acid residues 596-922 ofthe amino acid sequence shown in SEQ ID NO: 1. Domain C is composedentirely of beta-strands which form a single 8-stranded sheet structurethat folds back on itself, and thus may be described as a beta-sandwichstructure. One part of the beta-sheet forms the interface to Domain A.

[0060] Substrate Binding Site

[0061] Parts of the loop discussed above in the context of domains A, Band N2 are of particular interest for substrate interaction and activesite reactivity. In particular, in domain A, residues 439-443 in loop 1,residues 490-514 in loop 2, residues 621-628 in loop 4, residues 652-668in loop 5, residues 679-694 in loop 6, residues 733-740 in loop 7 andresidues 787-796 in loop 8; in domain B, residues 553-564 and 581-592 inloop 3; in domain N2, residues 400-404, wherein residue positionscorrespond to the amino acids in the amino acid sequence in SEQ ID NO:1.

[0062] Homology Building of Bacillus d ramificans Pullulanase or OtherPullulanases.

[0063] The structure of the Bacillus deramificans pullulanase (SEQ IDNO: 3) was model built on the structure disclosed in Appendix 1 herein.The structure of other pullulanases may be built analogously.

[0064] A model structure of a pullulanase can be built using theHomology program or a comparable program, e.g., Modeller (both fromMolecular Simulations, Inc., San Diego, Calif.). The principle is toalign the sequence of the pullulanase with the known structure with thatof the pullulanase for which a model structure is to be constructed. Thestructurally conserved regions can then be built on the basis ofconsensus sequences. In areas lacking homology, loop structures can beinserted, or sequences can be deleted with subsequent bonding of thenecessary residues using, e.g., the program Homology. Subsequentrelaxing and optimization of the structure should be done using eitherHomology or another molecular simulation program, e.g., CHARMm fromMolecular Simulations.

[0065] Methods for Designing Novel Pullulanase Variants

[0066] In a first aspect the present invention relates to a method forproducing a variant of a parent pullulanase, wherein the variant has atleast one altered property as compared to the parent pullulanase, themethod comprising:

[0067] i) modeling the parent pullulanase on the three-dimensionalstructure of SEQ ID NO: 1 depicted in Appendix 1 to produce athree-dimensional structure of the parent pullulanase;

[0068] ii) identifying in the three-dimensional structure obtained instep (i) at least one structural part of the parent pullulanase, whereinan alteration in the structural part is predicted to result in analtered property;

[0069] iii) modifying the nucleic acid sequence encoding the parentpullulanase to produce a nucleic acid sequence encoding a deletion,insertion, or substitution of one or more amino acids at a positioncorresponding to the structural part; and

[0070] iv) expressing the modified nucleic acid sequence in a host cellto produce the variant pullulanase.

[0071] Structural Part

[0072] The structural parts which is identified in step ii) of themethod of the invention may be composed of one amino acid residue.Normally, however, the structural part comprises more than one aminoacid residue, typically constituting one of the above-mentioned parts ofthe pullulanase structure such as one of the N1, N2, A, B, or C domains,an interface between any of these domains, a calcium binding site, aloop structure, the substrate binding site, or the like.

[0073] The structural or functional considerations may involve ananalysis of the relevant structure or structural part and itscontemplated impact on the function of the enzyme. For example, ananalysis of the functional differences between pullulanases and thevarious isoamylases may be used for assigning certain properties ofPromozyme®or homologeous model builded structure to certain parts of thePromozyme® or homologeous model build structure or to contemplate suchrelationship. For instance, differences in the pattern or structure ofloops surrounding the active site may result in differences in access tothe active site of the substrate and thus differences in substratespecificity and/or cleavage pattern.

[0074] Furthermore, parts of a pullulanase involved in substratebinding, and thus, for example, substrate specificity and/or cleavage,thermostability, and the like, have been identified (vide infra).

[0075] The modification of an amino acid residue or structural region istypically accomplished by suitable modifications of a nucleic acidsequence encoding the parent enzyme in question. The modification may besubstitution, deletion or insertion of an amino acid residue or astructural part.

[0076] The property to be modified may be stability (e.g.,thermostability), pH dependent activity, substrate specificity, such asdecreased condensation reactions, isoamylase like activity etc. Thus,the altered property may be an altered specific activity at a given pHand/or altered substrate specificity, such as an altered pattern ofsubstrate cleavage or an altered pattern of substrate inhibition.

[0077] In step ii) of the method according to the invention the part ofthe structure to be identified is preferably one which in the foldedenzyme is believed to be in contact with the substrate (cf. thedisclosure above in the section entitled “Substrate Binding Site”) orinvolved in substrate specificity and/or cleavage pattern, and/or onewhich is contributing to the pH or temperature profile of the enzyme, oris otherwise responsible for the properties of the pullulanase.

[0078] Described in the following are specific types of variants thathave been designed by use of the method of the invention.

[0079] The variants of the invention may comprise additionalmodifications in addition to the modifications described herein. Thevariants preferably have an amino acid sequence having more than 40%homology with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, preferablymore than 50%, e.g., more than 60%, such as more than 70%, more than75%, more than 80%, more than 85%, more than 90%, more than 91%, morethan 92%, more than 93%, more than 94%, more than 95%, more than 96%,more than 97%, more than 98% or more than 99% homology with the aminoacid sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.

[0080] In the present context the term “homologous to” or “homology”(also sometimes referred to as “similarity”) is used in it conventionalmeaning and the “homology” between two amino acid sequences may bedetermined by use of any conventional algorithm, preferably by use ofthe GAP program from the UWGCG package using default values for GAPpenalties, i.e. GAP creation penalty of 3.0 and GAP extension penalty of0.1 (Program Manual for the Wisconsin Package, Version 8, August 1994,Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711).The method is also described in S. B. Needleman and C. D. Wunsch,Journal of Molecular Biology, 48, 443-445 (1970).

[0081] Alternative Methods of Designing Pullulanase Variants

[0082] The three-dimensional structure of a pullulanase is influenced bythe presence (or absence) of the structural spatial location of ions,such as especially calcium ion (also sodium ions may be present). Suchinformation may be used as a parameter when designing a pullulanasevariant based on informations extracted from one parent pullulanase.

[0083] In one embodiment of the invention the parent pullulanase ischaracterized by having calcium ion(s) in the structure.

[0084] A specific example of such parent pullulanase is Klebsiella sp.mentioned above.

[0085] In another embodiment of the invention the parent pullulanase isa pullulanase which do not have metal ion(s) in the structure, inparticular calcium(s) ions.

[0086] A specific example of a parent pullulanase which do not havemetal ion(s) in the structure is PROMOZYME®

[0087] In general, informations in respect of structural parts (asdefined below) and/or structural elements, e.g., metal ions present inthe three-dimensional structure of a parent pullulanase, may be used foras a parameter when model building other corresponding parentpullulanases having, e.g., metal ion(s) in corresponding fixed spatiallylocation(s).

[0088] A parent pullulanase may have from no ions to one or more ions inthe structure, typically calcium and/or sodium ions. Parent pullulanaseshaving spatially similar located ions (as a solved three-dimensionalstructure) will also spatially have a similar three dimensionalstructure. This means that modeling of such a corresponding parentpullulanase with spatially fix ion(s) corresponding to that of, e.g.,Promozyme® (disclosed in Appendix 1), said corresponding parentpullulanase having at least 50% homology in the primary structure (asdefined above) when compared to SEQ ID NO: 1, 3, or 5, may according tothe invention be carried out as described below. If said correspondingparent pullulanase has at least 60%, or at least 70%, at least 80%, orat least 90% homology in the primary structure when compared to SEQ IDNO: 1, 3, or 5, the modeling will be even more precise.

[0089] Thus, in an aspect the invention relates to a method of producinga variant of a parent pullulanase having an altered property relative tothe parent pullulanase, wherein the parent pullulanase has the sequenceof SEQ ID Nos: 2, 4, 6, or has a sequence homology of at least 40% tothe sequence of SEQ ID NO: 2, 4, or 6, said method comprising

[0090] (a) identifying in the three-dimensional structure depicted inAppendix 1 at least one structural part wherein an alteration in acorresponding structural part in said parent pullulanase is predicted toresult in an altered property; wherein the altered property is selectedfrom the group consisting of thermostability, pH dependent activity,substrate specificity, isoamylase like activity, specific activity,altered pattern of substrate cleavage, or an altered pattern ofsubstrate inhibition;

[0091] (b) modifying the sequence of a nucleic acid encoding the parentpullulanase to produce a nucleic acid encoding a deletion, insertion, orsubstitution of one or more amino acids at a position corresponding tosaid structural part; and

[0092] (d) expressing the modified nucleic acid in a host cell toproduce the variant pullulanase;

[0093] wherein the variant has alpha-amylase enzymatic activity and hasat least one altered property relative to the parent.

[0094] Alternatively, the invention relates to a method of producing avariant of a parent pullulanase having an altered property relative tothe parent pullulanase, wherein the parent pullulanase has the sequenceof SEQ ID Nos: 2, 4, or 6, or has a sequence homology of at least 40% tothe sequence of SEQ ID NO: 2, 4, or 6, said method comprising

[0095] (a1) providing a three-dimensional pullulanase structure thatconsists of one or more of domains N1, N2, A, B, and C, in particularN1″;

[0096] (a2), do or do not contain one or more metal ions, in particularcalcium or sodium ion(s), in the structure;

[0097] (a3) a three dimensional structure corresponding/equivalent tothe three dimensional structure in shown in APPENDIX 1, one or more ofthe above mentioned domains;

[0098] (b) identifying in the three-dimensional structure at least onestructural part of the parent wherein an alteration in said structuralpart or corresponding structural part in said parent pullulanase ispredicted to result in said altered property and wherein said alteredproperty is selected from the group consisting of thermostability, pHdependent activity, substrate specificity, isoamylase like activity,specific activity, altered pattern of substrate cleavage, or an alteredpattern of substrate inhibition;

[0099] (c) modifying the sequence of a nucleic acid encoding the parentpullulanase to produce a nucleic acid encoding a deletion, insertion, orsubstitution of one or more amino acids at a position corresponding tosaid structural part; and

[0100] (d) expressing the modified nucleic acid in a host cell toproduce the variant alpha-amylase,

[0101] wherein the variant has alpha-amylase enzymatic activity and hasat least one altered property relative to the parent.

[0102] In an embodiment the parent pullulanase consists of domains N1″,N2, A, B, and C.

[0103] In an embodiment the parent pullulanase consists of domains N1′,N2, A, B, and C.

[0104] In an embodiment the parent pullulanase consists of domains N2,A, B, and C.

[0105] In an embodiment the parent pullulanase consists of domains N1′,N1″, N2, A, B, and C.

[0106] In an embodiment the parent pullulanase consists of domains N1″,N1′″, N2, A, B, and C.

[0107] Properties to be Modified

[0108] As mentioned above, the property to be modified may be stability(e.g., thermostability), pH dependent activity, substrate specificity,such as increased isoamylase activity, or specific activity. Thus, thealtered property may be an altered specific activity at a given pHand/or altered substrate specificity, such as an altered pattern ofsubstrate cleavage or an altered pattern of substrate inhibition.

[0109] In a particular interesting embodiment of the invention theproperty to be modified is the thermostability of the enzyme.

[0110] Common Structural Elements

[0111] Common structural elements referred to in vi) means elementsinvolved in keeping the three dimensional structure of the enzyme, e.g.,metal ions in the structure etc.

[0112] Thermostability

[0113] In the present context, the term “thermostable” (or“thermostability”) refers in general to the fact that the pullulanasevariants according to the invention have an improved thermostabilitycompared to the relevant parent pullulanase. The degree of improvementin thermostability can vary according to factors such as thethermostability of the parent pullulanase and the intended use of thepullulanase variant, i.e., whether it is primarily intended to be usedfor liquefaction or for saccharification or both. It will be apparentfrom the discussion below that for saccharification, the enzyme variantshould maintain a substantial degree of enzyme activity during thesaccharification step at a temperature of at least about 63° C.,preferably at least about 70° C., while an enzyme variant designed foruse in the liquefaction step should be able to maintain a substantialdegree of enzyme activity at a temperature of at least about 95° C.

[0114] The improved thermostability of enzyme variants according to theinvention can in particular be defined according to one or more of thefollowing criteria:

[0115] In one embodiment, the pullulanase variant of the invention hasan improved thermostability (and/or the method of the invention providesa pullulanase with an improved thermostabilty) as defined bydifferential scanning calorimetry (DSC) using the method describedherein.

[0116] In another embodiment, the pullulanase variant of the inventionhas an improved thermostability (and/or the method of the inventionprovides a pullulanase with an improved thermostabilty) as defined by anincreased half-time (T_(1/2)) of at least about 5%, preferably at leastabout 10%, more preferably at least about 15%, more preferably at leastabout 25%, most preferably at least about 50%, such as at least about100%, in the “T_(1/2) assay for liquefaction” described herein, using apH of 5.0 and a temperature of 95° C. Pullulanase variants according tothis definition are suitable for use in the liquefaction step of thestarch conversion process.

[0117] Alternatively or additionally, a pullulanase variant suitable foruse in liquefaction can be defined as having an improved thermostabilityas defined by an increased residual enzyme activity of at least about5%, preferably at least about 10%, more preferably at least about 15%,more preferably at least about 25%, most preferably at least about 50%,such as at least about 100%, in the “assay for residual activity afterliquefaction” described herein, using a pH of 5.0 and a temperature of95° C.

[0118] In a further embodiment, the enzyme variant of the invention hasan improved thermostability (and/or the method of the invention providesa pullulanase with an improved thermostabilty) as defined by anincreased half-time (T_(1/2)) of at least about 5%, preferably at leastabout 10%, more preferably at least about 15%, more preferably at leastabout 25%, most preferably at least about 50%, such as at least about100%, in the “T_(1/2) assay for saccharification” described herein,using a pH of 4.5 and a temperature of 70° C. Such variants are suitablefor use in the saccharification step of the starch conversion process.

[0119] Alternatively or additionally, a pullulanase variant suitable forsaccharification can be defined as having an improved thermostability asdefined by an increased residual enzyme activity of at least about 5%,preferably at least about 10%, more preferably at least about 15%, morepreferably at least about 25%, most preferably at least about 50%, suchas at least about 100%, in the “assay for residual activity aftersaccharification” described herein, using a pH of 4.5 and a temperatureof 63° C. Preferably, this improved thermostability is also observedwhen assayed at a temperature of 70° C.

[0120] The term “substantially active” as used herein for a givenpullulanase variant and a given set of conditions of temperature, pH andtime means that the relative enzymatic activity of the enzyme variant isat least about 25%, preferably at least about 50%, in particular atleast about 60%, especially at least about 70%, such as at least about90% or 95%, e.g., at least about 99% compared to the relative activityof the parent enzyme tested under the same set of conditions.

[0121] One advantage of the thermostable pullulanase of the invention isthat they make it possible to perform liquefaction and debranchingsimultaneously before the saccharification step. This has not previouslybeen possible, since the known pullulanases with acceptable specificactivity are thermolabile and are inactivated at temperatures above 60°C. (Some thermostable pullulanases from Pyrococcus are known, but thesehave an extremely low specific activity at higher temperatures and arethus unsuitable for purposes of the present invention). By debranching,using the thermostable pullulanases of the invention, duringliquefaction together with the action of an alpha-amylase, the formationof panose precursors is reduced, thereby reducing the panose content inthe final product and increasing the overall saccharification yield. Itis also possible in this manner to extend the liquefaction process timewithout risking formation of large amount of panose precursors. Byprolonging the liquefaction step, the DE yield is increased from 10-15to, e.g., 15-20, reducing the need for glucoamylase. This reducedglucoamylase requirement is in turn advantageous as the formation ofundesired isomaltose is reduced, thereby resulting in an increasedglucose yield. In addition, the reduced glucoamylase addition enablesthe saccharification step to be carried out at a higher substrateconcentration (higher DS, dry substances, concentration) than the normalapprox. 30-35% used according to the prior art. This allows reducedevaporation costs downstream, e.g., in a high fructose corn syrupprocess, and the saccharification reaction time can also be reduced,thereby increasing production capacity. A further advantage is thatalpha-amylase used in the liquefaction process does not need to beinactivated/denatured in this case.

[0122] Furthermore, it is also possible to use the thermostablepullulanases of the invention during saccharification, which isadvantageous for several reasons. In the conventional starchsaccharification process, the process temperature is not more than 60°C. due to the fact that neither the saccharification enzyme pullulanasenor AMG™ (i.e., glucoamylase) are sufficiently thermostable to allow theuse of a higher temperature. This is a disadvantage, however, as itwould be very desirable to run the process at a temperature of aboveabout 60° C., in particular above 63° C., e.g. about 70° C., to reducemicrobial growth during the relatively long saccharification step.Furthermore, a higher process temperature normally gives a higheractivity per mg of enzyme (higher specific activity), thereby making itpossible to reduce the weight amount of enzyme used and/or obtain ahigher total enzymatic activity. A higher temperature can also result ina higher dry matter content after saccharification, which would bebeneficial in terms of reducing evaporation costs.

[0123] In another interesting embodiment of the invention the propertyto be modified is the substrate specificity of the pullulanase, inparticular to modify the substrate specificity of the pullulanase insuch a way the variant pullulanase becomes more “isoamylase-like” in thesense of having an increased activity towards high molecular weightbranched starchy material such as glycogen and amylopectin. Methods fordetermining the substrate specificity of pullulanases are discussed inthe following section entitled “Methods for determining stability,activity and specificity”.

[0124] Thus, when used herein, the term “increased isoamylase activity”refers in general to the fact that the pullulanase variants according tothe invention exhibits a higher activity towards high molecular weightbranched starchy material, such as glycogen and amylopectin as comparedto the parent pullulanase.

[0125] The increased isoamylase activity of the pullulanase variantsaccording to the invention can in particular be defined according to thebelow criteria:

[0126] In one embodiment the pullulanase variant according to theinvention has an increased isoamylase activity as defined by an increaseof at least 5%, preferably of at least 10%, more preferably of at least15%, more preferably of at least 25%, most preferably of at least 50%,in particular of at least 75%, such as of at least 100% in the number ofreducing ends formed in the “assay for isoamylase-like activity”described herein, using 50 mM sodium acetate, a pH of 4.5, 5.0 or 5.5, atemperature of 60° C. and when incubated with a 10 w/v rabbit liverglycogen solution for a period of 10 min.

[0127] In the present context the term “pullulanase activity” isintended to mean that the pullulanase variant in question is capable ofdegrading pullulan when tested as described in the Examples (see thesection entitled “Determination of pullulanase activity).

[0128] Methods for Determining Stability, Activity and Specificity

[0129] Thermostability

[0130] Thermostability Assay 1

[0131] Thermostability of pullulanase variants of the invention may bedetected by measuring the residual activity by incubating the enzymeunder accelerated stress conditions, which comprise: pH 4.5 in a 50 mMsodium acetate buffer without a stabilizing dextrin matrix (such as theapproximately 35% dry matter which is normally present duringsaccharification). The stability can be determined at isotherms of,e.g., 63° C., 70° C., 80° C., 90° C. and 95° C., measuring the residualactivity of samples taken from a water bath at regular intervals (e.g.every 5 or 10 min.) during a time period of 1 hour. For determiningstability for the purpose of liquefaction, a pH of 5.0, a temperature of95° C. and a total assay time of 30 to 120 minutes are used (“assay forresidual activity after liquefaction”). For determining stability forthe purpose of saccharification, a pH of 4.5, a temperature of 63° C. or70° C. and a total assay time of 30 minutes are used (“assay forresidual activity after saccharification”).

[0132] Thermostability Assay 2 (T_(1/2))

[0133] Alternatively, the thermostability of pullulanase variants of theinvention may be expressed as a “half-time” (T_(1/2)), which is definedas the time, under a given set of conditions, at which the activity ofthe enzyme being assayed is reduced to 50% of the initial activity atthe beginning of the assay. In this case, the “T_(1/2) assay forliquefaction” uses a pH of 5.0 and a temperature of 95° C., while the“T_(1/2) assay for saccharification” uses a pH of 4.5 and a temperatureof 70° C. The assay is otherwise performed as described above for therespective assays for residual activity.

[0134] Activity: Somogyi-Nelson Method for Determination of ReducingSugars

[0135] The activity of pullulanases can be measured using theSomogyi-Nelson method for the determination of reducing sugars (J. Biol.Chem. 153, 375 (1944)). This method is based on the principle that sugarreduces cupric ions to cuprous oxide, which reacts with an arsenatemolybdate reagent to produce a blue colour that is measuredspectrophotometrically. The solution to be measured must contain 50-600mg of glucose per liter. The procedure for the Somogyi-Nelson method isas follows:

[0136] Sample value: Pipet 1 ml of sugar solution into a test tube. Add1 ml of copper reagent. Stopper the test tube with a glass bead. Placethe test tube in a boiling water bath for 20 minutes. Cool the testtube. Add 1 ml of Nelson's color reagent. Shake the test tube withoutinverting it. Add 10 ml of de-ionized water. Invert the test tube andshake vigorously. Measure the absorbance at 520 nm, inverting the testtube once immediately prior to transfer of the liquid to the cuvette.

[0137] Blank value: Same procedure as for the sample value, but withwater instead of sugar solution.

[0138] Standard value: Same procedure as for the sample value.Calculations: In the region 0-2 the absorbance is proportional to theamount of sugar. $\begin{matrix}{{{mg}\quad {sugar}\text{/}l} = \frac{100\quad \left( {{sample} - {blank}} \right)}{\left( {{standard} - {blank}} \right)}} \\{{\% \quad {glucose}} = \frac{\left( {{sample} - {blank}} \right)}{100 \times \left( {{standard} - {blank}} \right)}}\end{matrix}$

[0139] Reagents:

[0140] 1. Somogyi's Copper Reagent

[0141] 35.1 g Na₂HPO₄₀.2H₂O and 40.0 g potassium sodium tartrate(KNaC₄H₄O₂.4H₂O) are dissolved in 700 ml of de-ionized water. 100 ml of1N sodium hydroxide and 80 ml of 10% cupric sulphate (CuSO₄.5H₂O) areadded. 180 g of anhydrous sodium sulphate are dissolved in the mixture,and the volume is brought to 1 liter with de-ionized water.

[0142] 2. Nelson's Color Reagent

[0143] 50 g of ammonium molybdate are dissolved in 900 ml of deionizedwater. Then 42 ml of concentrated sulphuric acid are added, followed by6 g of disodium hydrogen arsenate heptahydrate dissolved in 50 ml ofdeionized water, and the volume is brought to 1 litre with deionizedwater. The solution is allowed to stand for 24-48 hours at 37° C. beforeuse and is stored in the dark in a brown glass bottle with a glassstopper.

[0144] 3. Standard

[0145] 100 mg of glucose (anhydrous) are dissolved in 1 liter ofde-ionized water.

[0146] Alternatively, the release of reducing sugars can be measuredusing a 96 well plate set-up modified after Fox, J. D. & Robyt, J. F.(1991) Anal. Biochem. 195,93-96. Assay conditions are (in brief): 1 mlsubstrate (e.g. 1% solution) in 50 mM citric acid pH 5 is preincubatedat 60° C. A zero timepoint is taken 150 micro 1 sample and transferredto a microtiter plate well containing 150 micro 1 solution A+B forreducing sugar determination. The enzymatic reaction is initiated byaddition of 100 micro 1 enzyme and time points are taken at T=1, 2, 3,4, and 5 min.

[0147] After completion of the assay, the plate is developed byincubation at 85° C. for 70 minutes and the plate is read at 540 nm.

[0148] Reagents for determination of reducing value: Solution A) andsolution B (62 mg copper sulfate pentahydrate and 63 mg L-serine in 50ml water).

[0149] Pullulanase Specificity

[0150] Methods for the determination and characterization of the profileof action and specificity of pullulanases for various substrates (e.g.,amylopectin, glycogen and pullulan) are described by Kainuma et al. inCarbohydrate Research, 61 345-357 (1978). Using these methods, therelative activity of a pullulanase can be determined, and the relativeactivity of a pullulanase variant according to the invention compared tothe relative activity of the parent pullulanase can be assessed, forexample to determine whether a pullulanase variant has the desiredincreased specificity toward high molecular weight saccharides, such asamylopectin, compared to the parent pullulanase.

[0151] In order to determine whether the pulluanase variant possesses anincreased isoamylase activity as compared to the parent pullulanse thefollowing test may be performed (“assay for isoamylase-like activity”):

[0152] 1000 mg rabbit liver glycogen is dissolved in 40 ml water towhich 0.2% NaOH has been added. 800 mg NaBH₄ is added carefully understirring. The solution is stirred for 48 hours at 25° C. after which thereaction is stopped by addition of Amberlite IR-118H (a cation exchangerwhich removes the boron ions and hence stops the reaction). The solutionis filtered to remove the matrix and evaporated to give 10 ml. Thesolution is then dialyzed extensively against de-ionized water to removeresidual boron ions. The parent pullulanase and the pulluanase variantare assayed according to the method of Somogyi-Nelson, using 50 mMsodium acetate, pH values of 4.5, 5.0 or 5.5 and a temperature of 60°C., with a reaction time of 10 minutes. Glucose is used as a standard, astandard curve being made from solutions containing of 0-200 mgglucose/liter.

[0153] Clearly, the higher the number of reducing ends formed during theincubation period, the higher “isoamylase activity”. The increase in thepullulanase variant's isoamylase activity is expressed as a percentagevalue based on the original “isoamylase activity” of the parentpullulanase.

[0154] Pullulanas Variants with Altered Stability

[0155] A variant with improved stability (typically increasedthermostability) may be obtained by substitution with proline,substitution of histidine with another amino acid, introduction of adisulfide bond, removal of a deamidation site, altering a hydrogen bondcontact, filling in an internal structural cavity with one or more aminoacids with bulkier side groups, introduction of interdomaininteractions, altering charge distribution, helix capping, orintroduction of a salt bridge.

[0156] Increased Mobility Regions:

[0157] The following regions have an increased mobility in the crystalstructure of Promozyme®, and it is presently believed that these regionscan be responsible for stability or activity of the enzyme. Improvementsof the enzyme can be obtained by mutation in the below regions andpositions. Introducing e.g. larger residues or residues having moreatoms in the side chain could increase the stability, or, e.g.,introduction of residues having fewer atoms in the side chain could beimportant for the mobility and thus the activity profile of the enzyme.The regions can be found by analysing the B-factors taken from thecoordinate file in Appendix 1, and/or from molecular dynamicscalculations of the isotropic fluctuations. These can be obtained byusing the program CHARMm from MSI (Molecular simulations inc.).

[0158] Thus, in order to stabilize mobile regions in the structure, apreferred variant of a parent pullulanase comprises a modification,e.g., a substitution, of an amino acid residue corresponding to one ormore of the following residues of the amino acid sequence set forth inSEQ ID NO: 1:

[0159] 408-429 (i.e., 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 and 429),

[0160] 300-314 (i.e., 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,310, 311, 312, 313 and 314),

[0161] 157-165 (i.e., 157, 158, 159, 160, 161, 162, 163, 164 and 165),95-113 (i.e., 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112 and 113),

[0162] 130-140 (i.e., 130, 131, 132, 133, 134, 135, 136, 137, 138, 139and 140),

[0163] 232-238 (i.e., 232, 233, 234, 235, 236, 237 and 238),

[0164] 266-272 (i.e., 266, 267, 268, 269, 270, 271 and 272),

[0165] 302-308 (i.e., 302, 303, 304, 305, 306, 307 and 308),

[0166] 418-428 (i.e., 418, 419, 420, 421, 422, 423, 424, 425, 426, 427and 428),

[0167] 500-507 (i.e., 500, 501, 502, 503, 504, 505, 506 and 507),

[0168] 659-665 (i.e., 659, 660, 661, 662, 663, 664 and 665) and

[0169] 751-755 (i.e., 751, 752, 753, 754 and 755).

[0170] Similar modifications, e.g., substitutions, may be introduced inequivalent positions of other pullulanases. Variants of particularinterest have a combination of one or more of the above with any of theother modifications disclosed herein.

[0171] For example, other preferred modifications, e.g., substitutions,which are believed to stabilized mobile regions in the structure of thepullulanase from Bacillus deramificans, correspond to one or more of thefollowing residues of the amino acid sequence set forth in SEQ ID NO: 3:

[0172] 406-427 (i.e., 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426 and 427),

[0173] 298-312 (i.e., 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,308, 309, 310, 311 and 312),

[0174] 153-161 (i.e., 153, 154, 155, 156, 157, 158, 159, 160 and 161),

[0175] 91-109 (i.e., 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108 and 109),

[0176] 126-136 (i.e., 126, 127, 128, 129, 130, 131, 132, 133, 134, 135and 136),

[0177] 230-236 (i.e., 230, 231, 232, 233, 234, 235 and 236),

[0178] 264-270 (i.e., 264, 265, 266, 267, 268, 269 and 270),

[0179] 300-306 (i.e., 300, 301, 302, 303, 304, 305 and 306),

[0180] 416-426 (i.e., 416, 417, 418, 419, 420, 421, 422, 423, 424, 425and 426),

[0181] 498-505 (498, 499, 500, 501, 502, 503, 504 and 505),

[0182] 656-662 (i.e., 656, 657, 658, 659, 660, 661 and 662) and

[0183] 749-753 (i.e., 749, 750, 751, 752 and 753).

[0184] Contemplated combinations of substitutions include according tothe invention one or more of: deletion (1-111); deletion (1-113); D562P;G292P; G794P; D148P; N119P; N400S; N400L; N400F; N446S; N446L; N446F;N504S; N504L; N₅O₄F; N735S; N735L; N735F; N789S; N789L; N789F; I566A;Q485H; V551I; S492F; D444R; D444K; deletion (154-273).

[0185] Multi substitutions of the invention include: deletion(1-111)+D562P; deletion (1-111)+G292P; deletion (1-111)+G794P; deletion(1-111)+D148P; deletion (1-111)+N119P+N400S/L/F; deletion(1-111)+N446L/F; deletion (1-111)+N₅O₄S/L/F; deletion (1-111)+N735S/L/F;deletion (1-111)+N789S/L/F; deletion (1-111)+1566A; deletion(1-111)+Q485H; deletion (1-111)+V551I; deletion (1-111)+S492F; eletion(1-111)+D444R/K; eletion (1-111)+deletion (154-273); deletion(1-113)+D562P; deletion (1-111)+G292P; eletion (1-113)+G794P; deletion(1-113)+D148P; deletion (1-113)+N119P+N40OS/L/F; deletion(1-131)+N446L/F; eletion (1-113)+N₅O₄S/L/F; deletion (1-113)+N735S/L/F;eletion (1-113)+N789S/L/F; deletion (1-113)+1566A; deletion(1-113)+Q485H; deletion (1-113)+V551I; deletion (1-113)+S492F; eletion(1-113)+D444R/K; deletion (1-113)+deletion (154-273); D562P+G292P;D562P+G794P; D562P+D148P; D562P+N119P; D562P+N400S/L/F; D562P+N446S/L/F;D562P+N₅O₄S/L/F; D562P+N735S/L/F; D562P+N789S/L/F; D562P+1566A;D562P+Q485H; D562P+V551I; D562P+S492F; D562P+D444R/K; D562P+deletion(154-273); G292P+G794P; G292P+D148P; G292P+N119P; G292P+N400S/L/F;G292P+N446S/L/F; G292P+N₅O₄S/L/F; G292P+N735S/L/F; G292P+N789S/L/F;G292P+1566A; G292P+Q485H; G292P+V551I; G292P+S492F; G292P+D444R/K;G292P+deletion (154-273); G794P+D148P; G794P+N119P; G794P+N400S/L/F;G794P+N446S/L/F; G794P+N₅O₄S/L/F; G794P+N735S/L/F; G794P+N789S/L/F;G794P+1566A; G794P+Q485H; G794P+V551I; G794P+S492F; G794P+D444R/K;G794P+deletion (154-273); 148P+N119P; D148P+N400S/L/F; D148P+N446S/L/F;D148P+N₅O₄S/L/F; D148P+N735S/L/F; D148P+N789S/L/F; D148P+1566A;D148P+Q485H; D148P+V551I; D148P+S492F; D148P+D444R/K; D148P+deletion(154-273); N119P+N400S/L/F; N119P+N446S/L/F; N119P+N₅O₄S/L/F;N119P+N735S/L/F; N119P+N789S/L/F; N119P+1566A; N119P+Q485H; N119P+V551I;N119P+S492F; N119P+D444R/K; N119P+deletion (154-273);N400S/L/F+N446S/L/F; N400S/L/F+N₅O₄S/L/F; N400S/L/F+N735S/L/F;N400S/L/F+N789S/L/F; N400S/L/F+1566A; N400S/L/F+Q485H; N400S/L/F+V551I;N400S/L/F+S492F; N400S/L/F+D444R/K; N400S/L/F+deletion (154-273);N446S/L/F+N₅O₄S/L/F; N446S/L/F+N735S/L/F; N446S/L/F+N789S/L/F;N446S/L/F+1566A; N446S/L/F+Q485H; N446S/L/F+V551I; N446S/L/F+S492F;N446S/L/F+D444R/K; N446S/L/F+deletion (154-273); N₅O₄S/L/F+N735S/L/F;N₅O₄S/L/F+N789S/L/F; N₅O₄S/L/F+1566A; N₅O₄S/L/F+Q485H; N₅O₄S/L/F+V551I;N₅O₄S/L/F+S492F; N₅O₄S/L/F+D444R/K; N₅O₄S/L/F+deletion (154-273);N735S/L/F+N789S/L/F; N735S/L/F+1566A; N735S/L/F+Q485H; N735S/L/F+V551I;N735S/L/F+S492F; N735S/L/F+D444R/K; N735S/L/F+deletion (154-273);N789S/L/F+1566A; N789S/L/F+Q485H; N789S/L/F+V551I; N789S/L/F+S492F;N789S/L/F+D444R/K; N789S/L/F+deletion (154-273) I566A+Q485H;1566A+V551I; 1566A+S492F; 1566A+D444R/K; 1566A+deletion (154-273)Q485H+V551I; Q485H+S492F; Q485H+D444R/K; Q485H+deletion (154-273);V551I+S492F; V551I+D444R/K; V551I+deletion (154-273); S492F+D444R/K;S492F+deletion (154-273); D444R/K⁺ deletion (154-273); deletion(1-111)+D562P+N400S/L/F; deletion (1-111)+G292P+N400S/L/F; deletion(1-111)+G794P+N400S/L/F; deletion (1-111)+D148P+N400S/L/F; deletion(1-111)+N119P+N40OS/L/F; deletion (1-111)+N446L/F+N400S/L/F; deletion(1-111)+N₅O₄S/L/F+N400S/L/F; deletion (1-111)+N735S/L/F+N400S/L/F;deletion (1-111)+N789S/L/F+N400S/L/F; deletion (1-111)+1566A+N400S/L/F;deletion (1-111)+Q485H+N400S/L/F; deletion (1-111)+V551I+N400S/L/F;deletion (1-111)+S492F+N400S/L/F; deletion (1-111)+D444R/K⁺ N400S/L/F;deletion (1-111)+deletion (154-273)+N400S/L/F; deletion (1-111)+D562P;deletion (1-111)+G292P; eletion (1-111)+G794P; deletion (1-111)+D148P;deletion (1-111)+N119P+N400S/L/F; deletion (1-111)+N446L/F; deletion(1-111)+N₅O₄S/L/F; deletion (1-111)+N735S/L/F; deletion(1-111)+N789S/L/F; deletion (1-111)+1566A; deletion (1-111)+Q485H;deletion (1-111)+V551I;

[0186] Furthermore, it is envisaged from the structure that deletion ofcertain amino acid residues will confer increased stability, such asincreased thermostability, to the thus produced variant. Variants, whichare believed to be of particular importance, comprises a deletion ofamino acid residues corresponding to the following residues of the aminoacid sequence set forth in SEQ ID NO: 1:

[0187] Deletion of the peptide fragment 158-275, such as a deletionstarting from position 158, 159, 160 or 161 and ending at position 270,271, 272, 273, 274 or 275, i.e., the longest deletion will be deletionof the peptide fragment 158-275 and the shortest deletion will bedeletion of the peptide fragment 161-270.

[0188] Other deletions which are expected to confer increased stability,such as increased thermostability, to the pullulanase variant comprisesa deletion of amino acid residues corresponding to the followingresidues of the amino acid sequence set forth in SEQ ID NO: 1:

[0189] Deletion of the peptide fragment 1-315, such as deletion of thepeptide fragment 1-314, 1-313, 1-312, 1-311, 1-310, 1-309, 1-308, 1-307,1-306, 1-305, or 1-304.

[0190] Furthermore, the following deletions are expected to conferincreased stability, such as increased thermostability, to thepullulanase variant comprises a deletion of amino acid residuescorresponding to the following residues of the amino acid sequence setforth in SEQ ID NO: 1:

[0191] Deletion of the peptide fragment 1-115, such as deletion of thepeptide fragment 1-114, 1-113, 1-112, 1-111, 1-110, 1-109, 1-108, 1-107,1-106 or 1-105.

[0192] Similar deletions may be introduced in equivalent positions ofother pullulanases. Variants of particular interest have a combinationof one or more of the above with any of the other modificationsdisclosed herein.

[0193] For example, it is envisaged that deletion of the below aminoacid residues will confer increased stability, such as increasedthermostability, to the thus produced variant of the pullulanase fromBacillus deramificans (SEQ ID NO: 3):

[0194] Deletion of the peptide fragment 154-273, such as a deletionstarting from position 154, 155, 156 or 157 and ending at position 268,269, 270, 271, 272 or 273, i.e. the longest deletion will be deletion ofthe peptide fragment 154-273 and the shortest deletion will be deletionof the peptide fragment 157-268.

[0195] Other deletions which are expected to confer increased stability,such as increased thermostability, to the pullulanase variant comprisesa deletion of amino acid residues corresponding to the followingresidues of the amino acid sequence set forth in SEQ ID NO: 3:

[0196] Deletion of the peptide fragment 1-313, such as deletion of thepeptide fragment 1-312, 1-311, 1-310, 1-309, 1-308, 1-307, 1-306, 1-305,1-304, or 1-303.

[0197] Furthermore, the following deletions are expected to conferincreased stability, such as increased thermostability, to thepullulanase variant comprises a deletion of amino acid residuescorresponding to the following residues of the amino acid sequence setforth in SEQ ID NO: 3:

[0198] Deletion of the peptide fragment 1-111, such as deletion of thepeptide fragment 1-113, 1-111, 1-110, 1-109, 1-108, 1-107, 1-106, 1-105,1-104, 1-103, 1-102 or 1-101.

[0199] Cavities and Crevices

[0200] The structure of the pullulanase contains a number of uniqueinternal cavities, which may contain water, and a number of crevices. Inorder to increase the stability, preferably the thermostability, of thepullulanase it may be desirable to reduce the number or size of cavitiesand crevices, e.g., by introducing one or more hydrophobic contacts,preferably achieved by introducing amino acids with bulkier side chainsin the vicinity or surroundings of the cavity or crevice. For instance,the amino acid residues to be modified are those that are involved inthe formation of a cavity or crevice.

[0201] In order to determine which amino acid residues of a given enzymeare involved in the formation of cavities or crevices the Conollyprogram is normally used (B. Lee and F. M. Richards, J. Mol. Biol. 55,379-400 (1971)). The program uses a probe with a certain radius tosearch the external and internal surface of the protein. The smallestcrevice observable in this way has the probe radius.

[0202] To analyze the solved structure of Promozyme®, a modified versionof the Connolly program included in the program of INSIGHT was used. Inthe first step, the water molecules and the ions were removed byunmerging these atoms from the solved structure. By using the commandMOLECULE SURFACE SOLVENT the solvent accessible surface area wascalculated for all atoms and residues using a probe radius of 1.4 Å, anddisplayed graphically together with the model of the solved structure.The internal cavities are then seen as dot surfaces with no connectionsto the external surface.

[0203] Suggestions for specific modifications to fill out the cavitiesare given below. By using the homology built structures and/orcomparisons based on sequence alignment, mutations for homologousstructures of pullulanases can be made.

[0204] Accordingly, in a further aspect the present invention relates toa method for constructing a variant of a parent pullulanase, the methodcomprising:

[0205] a) identifying an internal cavity or crevice in thethree-dimensional structure of the parent pullulanase;

[0206] b) substituting at least one amino acid residue involved in theformation of a cavity or crevice with another amino acid residue whichincreases the hydrophobic interaction and/or fills out or reduces thesize of the cavity or crevice;

[0207] c) optionally repeating steps a) and b) recursively;

[0208] d) optionally, making alterations each of which is an insertion,a deletion or a substitution of an amino acid residue at one or morepositions other than b);

[0209] e) preparing the variant resulting from steps a)-d);

[0210] f) testing the stability and/or the temperature dependentactivity profile of the variant; and

[0211] g) optionally repeating steps a)-f) recursively; and

[0212] h) selecting a variant having increased stability and/or analtered temperature dependent activity profile as compared to the parentpullulanase.

[0213] In a preferred embodiment of the invention the variant pullanaseprovided by the above method have increased thermostability as comparedto the parent pullulanase. The thermostability of a given variant may beassessed as described in the above section entitled “Methods fordetermining stability, activity and specificity”.

[0214] It will be understood that the cavity or crevice is identified bythe amino acid residues surrounding said cavity or crevice, and thatmodification of said amino acid residues are of importance for fillingor reducing the size of the cavity or crevice. Preferably, themodification is a substitution with a bulkier amino acid residue, i.e.one with a greater side chain volume or with an increased number ofatoms in the side chain. For example, all the amino acids are bulkierthan Gly, whereas Tyr and Trp are bulkier than Phe. The particular aminoacid residues referred to below are those that in a crystal structurehave been found to flank the cavity or crevice in question.

[0215] In a preferred embodiment, the variant of a pullulanase, in orderto fill, either completely or partly, cavities or crevices locatedinternally or externally in the structure, comprises a modification,e.g., a substitution, of an amino acid residue corresponding to one ormore of the following residues of the amino acid sequence set forth inSEQ ID NO: 1: 406, 394, 568, 573 576, 563, 557, 396, 392, 515, 583, 442,792, 767, 732, 760, 783, 740, 688, 478, 534, 550, 627, 314.

[0216] In a more preferred embodiment, the variant of a pullulansecomprises one or more substitutions corresponding to the followingsubstitutions in the amino acid sequence set forth in SEQ ID NO: 1:G406A, P394F/W/I/L, 1568L/F, Y573W, T576N/L/I, S563T, T557N, A396V/L/I,V392, N515M/L/I, V583I/F/L, D442Q, S792Y/F, V767Q/E/L/I, V732I/L,D760Q/E/F/Y, L783F/Y, L740Q, D688Y/F/E/Q/R/K, L478Q/R, L534F/Y/I,M550F/Y/I/L, L627F/Y/I, L314I.

[0217] Similar modifications, e.g., substitutions, may be introduced inequivalent positions of other pullulanases. Variants of particularinterest have a combination of one or more of the above with any of theother modifications disclosed herein.

[0218] For example, the variant of a pullulanase may also comprise oneor more substitutions corresponding to the following substitutions inthe amino acid sequence set forth in SEQ ID NO: 3: 566, 485, 487, 437,775, 779, 551, 428, 492, 495, 392, 621, 437+503, 674+664 and 823.

[0219] In a more preferred embodiment, the variant of a pullulansecomprises one or more substitutions corresponding to the followingsubstitutions in the amino acid sequence set forth in SEQ ID NO: 3:1566A, Q485H, M487L, D437H, Q775H, E779D, V5511, 1428Y/F, S492F,V495I/F/Y, P392Y, L621Q, D437H+D503Y, V674+L664F and L823V.

[0220] Disulfide Bonds

[0221] A variant with improved stability (typically improvedthermostability) as compared to the parent pullulanase may be obtainedby introducing new interdomain and intradomain contacts, such asestablishing inter- or intradomain disulfide bridges.

[0222] Accordingly, a further aspect of the present invention relates toa method for constructing a variant of a parent pullulanase, the methodcomprising:

[0223] a) identifying in the three-dimensional structure of the parentpullulanase two or more amino acid residues which, when substituted withcysteines, are capable of forming a disulfide bond;

[0224] b) substituting the amino acids identified in a) with cysteines;

[0225] c) optionally repeating steps a) and b) recursively;

[0226] d) optionally, making alterations each of which is an insertion,a deletion or a substitution of an amino acid residue at one or morepositions other than b);

[0227] e) preparing the variant resulting from steps a)-d);

[0228] f) testing the stability of said variant; and

[0229] g) optionally repeating steps a)-f) recursively; and

[0230] h) selecting a variant having increased stability as compared tothe parent pullulanase.

[0231] In a preferred embodiment of the invention the variant pullanaseprovided by the above method have increased thermostability as comparedto the parent pullulanase. The thermostability of a given variant may beassessed as described in the above section entitled “Methods fordetermining stability, activity and specificity”.

[0232] In order to determine, in the three-dimensional structure of theparent pullulanase, the amino acid residues which, when substituted withcysteines, are capable of forming a disulfide bond, residues with CBatoms less than 4A from each other, and where the direction of the CA-CBfrom each residue is pointing towards the other residue are identified.Following the above-mentioned guidelines, the below amino acid residueswere identified in the amino acid sequence of SEQ ID NO: 1, and it iscontemplated that these residues are suitable for cystein replacement,thereby opening up the possibility of establishing one or more disulfidebridges in the variant pullulanase: K758C+I914C, T916C+A765C,1897C+S819C, P525C+E499C, H286C+T148C.

[0233] Similar substitutions may be introduced in equivalent positionsof other pullulanases. Variants of particular interest have acombination of one or more of the above with any of the othermodifications disclosed herein.

[0234] For example, it is contemplated that the following residues,identified in the amino acid sequence of the pullulanase from Bacillusderamificans (SEQ ID NO: 3), are suitable for cystein replacement,thereby opening up the possibility of establishing one or more disulfidebridges in the variant pullulanase: K756C/I912C, M914C/A763C,V895C/G817C, A523C/E497C, H284C/T144C.

[0235] Surface Charge Distribution

[0236] A variant with improved stability (typically improvedthermostability) as compared to the parent pullulanase may be obtainedby changing the surface charge distribution of the pullulanase. Forexample, when the pH is lowered to about 5 or below histidine residuestypically become positively charged and, consequently, unfavorableelectrostatic interactions on the protein surface may occur. Byengineering the surface charge of the pullulanase one may avoid suchunfavorable electrostatic interactions that in turn leads to a higherstability of the pullulanase.

[0237] Therefore, a further aspect of the present invention relates tomethod for constructing a variant of a parent pullulanase, the methodcomprising:

[0238] a) identifying, on the surface of the parent pullulanase, atleast one amino acid residue selected from the group consisting of Asp,Glu, Arg, Lys and His;

[0239] b) substituting, on the surface of the parent pullulanase, atleast one amino acid residue selected from the group consisting of Asp,Glu, Arg, Lys and His with an uncharged amino acid residue;

[0240] c) optionally repeating steps a) and b) recursively;

[0241] d) optionally, making alterations each of which is an insertion,a deletion or a substitution of an amino acid residue at one or morepositions other than b);

[0242] e) preparing the variant resulting from steps a)-d);

[0243] f) testing the stability of said variant; and

[0244] g) optionally repeating steps a)-f) recursively; and

[0245] h) selecting a variant having increased stability as compared tothe parent pullulanase.

[0246] As will be understood by the skilled person it may also, in somecases, be advantageous to substitute an uncharged amino acid residuewith an amino acid residue bearing a charge or, alternatively, it may insome cases be advantageous to substitute an amino acid residue bearing acharge with an amino acid residue bearing a charge of opposite sign.Thus, the above-mentioned method may easily be employed by the skilledperson also for these purposes. In the case of substituting an unchargedamino acid residue with an amino acid residue bearing a charge theabove-mentioned method may be employed the only difference being stepsa) and b) which will then read:

[0247] a) identifying, on the surface of the parent pullulanase, atleast one uncharged amino acid residue;

[0248] b) substituting, on the surface of the parent pullulanase, atleast one uncharged amino acid residue with a charged amino acid residueselected from the group consisting of Asp, Glu, Arg, Lys and His.

[0249] Also in the case of changing the sign of an amino acid residuepresent on the surface of the pullulanase the above method may beemployed. Again, compared to the above method, the only difference beingsteps a) and b) which, in this case, read:

[0250] a) identifying, on the surface of the parent pullulanase, atleast one charged amino acid residue selected from the group consistingof Asp, Glu, Arg, Lys and His;

[0251] b) substituting, on the surface of the parent pullulanase, atleast one charged amino acid residue identified in step a) with an aminoacid residue having an opposite charge.

[0252] Thus, Asp may be substituted with Arg, Lys or His; Glu may besubstituted with Arg, Lys or His; Arg may be substituted with Asp orGlu; Lys may be substituted with Asp or Glu; and His may be substitutedwith Asp or Glu.

[0253] In a preferred embodiment of the invention the variantpullulanase provided by the above method(s) have increasedthermostability as compared to the parent pullulanase. Thethermostability of a given variant may be assessed as described in theabove section entitled “Methods for determining stability, activity andspecificity”.

[0254] In order to determine the amino acid residues of a pullulanase,which are present on the surface of the enzyme, the surface accessiblearea are measured using the DSSP program (Kabsch and Sander, Biopolymers(1983), 22, 2577-2637). All residues having a surface accessibiltyhigher than 0 is regarded a surface residue.

[0255] The amino acid residues found on the surface of Promozyme® usingthe above method are as follows: E526, Q544, E760, N338, N228, N181,

[0256] and it is contemplated that the following substitutions are ofparticular interest: E526H, Q544E, E760Q, N338K/R, N228DE/, N181K/R.

[0257] Similar substitutions may be introduced in equivalent positionsof other pullulanases. Variants of particular interest have acombination of one or more of the above with any of the othermodifications disclosed herein.

[0258] For example, the variant of a pullulanase may also comprise oneor more modifications, e.g., substitutions, corresponding to thefollowing substitutions in the amino acid sequence set forth in SEQ IDNO: 3: 444, 530, 710 and 855.

[0259] In a more preferred embodiment, the variant of a pullulansecomprises one or more substitutions corresponding to the followingsubstitutions in the amino acid sequence set forth in SEQ ID NO: 3:D444R/K, K530Y/F/L, N710R and T855K.

[0260] Other Modifications

[0261] Variants with improved stability, in particular variants withimproved thermostability, can be obtained by improving existing orintroducing new interdomain or intradomain contacts. Such improvedstability can be achieved by the modifications listed below.

[0262] Thus, one preferred embodiment of the invention relates to avariant of a parent pullulanase which has an improved stability and oneor more salt bridges as compared to the parent pullulanase, wherein saidvariant comprises a modifications, e.g., a substitution, in a positioncorresponding to at least one of the following sets of positions in SEQID NO: 1: 301, 385, 298, 299, 385 and 299+385, in particular L301R,N385R, H298R, N299R, N385D and N299R+N385D.

[0263] Similar modifications, e.g., substitutions, may be introduced inequivalent positions of other pullulanases. Variants of particularinterest have a combination of one or more of the above with any of theother modifications disclosed herein.

[0264] For example, it is contemplated that the following substitutionsin the pullunanase having the amino acid sequence set forth in SEQ IDNO: 3 will enhance the stability of the enzyme: T891D, S892K,T891D+S892K and N400R.

[0265] In another preferred embodiment, the variant of the pullulanasecomprises a substitution corresponding to one or more of the followingsubstitutions with proline in the amino acid sequence set forth in SEQID NO: 1: G293P, K151P, K122P, N315P, N374P, N793P, A446P, G672P, G668P,T556P

[0266] In a further interesting embodiment of the invention, the variantof the pullulanase comprises a substitution corresponding to one or moreof the following substitutions with proline in the amino acid sequenceset forth in SEQ ID NO: 3: D562P, G794P, G292P, D148P, N119P, D314P,N373P, N792P, G671P, G667P, and T554P.

[0267] Analogously, it may be preferred that one or more histidineresidue(s) present in the parent pullulanase is (are) substituted with anon-histidine residues such as Y, V I, L, F, M, E, Q, N, or D.Accordingly, in another preferred embodiment, the variant of the parentpullulanase comprises a substitution of an amino acid residuecorresponding to one or more of the following residues of the amino acidsequence set forth in SEQ ID NO: 3: H422Y/F/L, H483Y/F/L, H543Y/F/L/Nand H613Y/F/L.

[0268] It may be preferred that one or more asparagine or glutamineresidues present in the parent pullulanase is or are substituted with aresidue lacking the amide group on the side chain. Preferably, suchasparagines or glutamine residues are substituted with S, T, V, L and/orF amino acid residues. Accordingly, in another preferred embodiment, thevariant of the parent pullulanase comprises a modification, e.g. asubstitution, of an amino acid residue corresponding to one or more ofthe following residues of the amino acid sequence set forth in SEQ IDNO: 1: Q543, Q339, N337, Q380, Q353, N384, N286, N298, N227, Q227, Q210,N180, Q259, N583, N790, N793, N₅O₅, N788, N736, N684, N689 or N681,preferably Q543S/T/V/L/F, Q339S/T/V/L/F, N337S/T/V/L/F, Q380S/T/V/L/F,Q353S/T/V/L/F, N384S/T/V/L/F, N286S/T/V/L/F, N298S/T/V/L/F,N227S/T/V/L/F, Q227S/T/V/L/F, Q210S/T/V/L/F, N180S/T/V/L/F,Q259S/T/V/L/F, N583S/T/V/L/F, N790S/T/V/L/F, N793S/T/V/L/F,N505S/T/V/L/F, N788S/T/V/L/F, N736S/T/V/L/F, N684S/T/V/L/F,N689S/T/V/L/F and N681S/T/V/L/F.

[0269] The corresponding residues found in the pullulanase from Bacillusderamificans (SEQ ID NO: 3) include: N400, N446, N₅O₄, N717, N735 andN789, preferably N400S/T/V/L/F, N446S/T/V/L/F, N₅O₄S/T/V/L/F,N717S/T/V/L/F, N735S/T/V/L/F, and N789S/T/V/L/F.

[0270] Moreover, it is contemplated that modifications, e.g.substitutions, in the region linking the N2 and the A domain, as well asother regions linking other domains, will confer additional stability,such as an increased thermostability, to the enzyme. Thus, in aninteresting embodiment of the invention, the pullulanase variantcomprises one or more modifications, e.g., substitutions, in thedomain-linking regions (e.g., the region linking the N2 and A domains).

[0271] Examples of such modifications include one or more of thefollowing substitutions in the pullulanase from Bacillus deramificans(SEQ ID NO: 3): 111, 112, 158-160 (i.e., 158, 159 and 160), 270-274(i.e., 270, 271, 272, 273 and 274), 302-314 (i.e., 302, 303, 304, 305,306, 307, 308, 309, 310, 311, 312, 313 and 314) and 408-426 (i.e., 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425 and 426).

[0272] Examples of specific substitutions are: S111T/V/L, N112S/T/Q,S158Y/F/T, L159Y/K/R/A/S/T, G160A/S/T, D270E/S/T, L271V/I, V272I,T273N/D/E/Y/F, V274I, N₃O₂V/L/Y, N₃O₅V/L/Y, S306T/V, Q308K/R/A/S/T,Y309F, Y310E/D/Q/N/L/V/I, D314A/S/T, L409N, D408S/T, A410S/T,D413R/K/S/T, A415S/T, G416S/T/V, N418A/V/S/T, S419D/N/T,K421E/Q/S/T/V/A, H422D/L/Y/F, 1423L/V/S/T/N/Q, T424S/A and K426A/S/T.

[0273] Other substitutions that are considered of particular importancein SEQ ID NO: 3 include D437N and D440N.

[0274] Similar modifications, e.g., substitutions, may be introduced inequivalent positions of other pullulanases. Modifications of particularinterest are any combination of one or more of the above with any of theother modifications disclosed herein.

[0275] Before actually constructing a pullulanase variant to achieve anyof the above objectives, it may be convenient to evaluate whether or notthe contemplated amino acid modification can be accommodated intopullulanase structure, e.g., in a model of the three-dimensionalstructure of the parent pullulanase.

[0276] Pullulanase Variants with Altered Substrate Specificity

[0277] One aim of the present invention is to change the degradationcharacteristics of a pullulanase. Thus, as Promozyme® (and pullulanasesin general) exhibits a low activity towards high molecular weightbranched starchy material, such as glycogen and amylopectin, it may bedesirable to change this cleavage pattern, e.g., so as to obtain ahigher activity against such substrates, in particular when thepullulanase is to be added during the liquefaction process.

[0278] An altered substrate specificity may be achieved by modifying thesubstrate binding area in a parent pullulanse.

[0279] Accordingly, the present invention also relates to a method forconstructing a variant of a parent pullulanase, the method comprising:

[0280] a) identifying the substrate binding area in a model of thethree-dimensional structure of the parent pullulanase;

[0281] b) modifying the substrate binding area by an amino acidsubstitution, deletion and/or insertion;

[0282] c) optionally repeating step b) recursively;

[0283] d) optionally, making alterations each of which is an insertion,a deletion or a substitution of an amino acid residue at one or morepositions other than b),

[0284] e) preparing the variant resulting from steps a)-d);

[0285] f) testing the substrate specificity of the variant;

[0286] g) optionally repeating steps a)-f) recursively; and

[0287] h) selecting a variant having an altered substrate specificity ascompared to the parent pullulanase.

[0288] The substrate binding area may easily be identified by homologyto other family 13 members. The active site residues are identified byhomology. The substrate-binding site is identified by the concave cavitycontaining the active site residues. A substrate model is docked intothe cavity. A suitable substrate model is the substrate structure foundin the pdb file 1BAG termed GLC. This model can be “docked” into thePromozyme X-ray structure or a modeled Pullulanase 3D structure bysuperimposing the active site residues in the two structures. In 1BAGone of the active site residues has been mutated into a Gln instead ofthe native Glu. The active site residues to be superimposed are: D269,Q208 and D176 (1BAG) with D736, E651 and D622 (Promozyme®). Thesuperposition can be made using the program INSIGHTII.

[0289] Without being limited to any theory, it is presently believedthat binding between a substrate and an enzyme is supported by favorableinteractions found within a sphere 10 Å from the substrate molecule, inparticular within a sphere of 6 Å from the substrate molecule. Examplesof such favorable bonds are hydrogen bonds, strong electrostaticinteraction and/or hydrophobic interactions. The following residues ofPromozyme® (SEQ ID NO: 1), are within a distance of 10 Å from the“docked” substrate and thus believed to be involved in interactions withsaid substrate:

[0290] 437, 439, 487, 489, 490, 514, 679, 681, 684, 685, 731, 775, 786,

[0291] 494-496 (i.e., 494, 495 and 496),

[0292] 505-511 (i.e., 505, 506, 507, 508, 509, 510 and 511),

[0293] 551-559 (i.e., 551, 552, 553, 554, 555, 556, 557, 558 and 559),

[0294] 584-590 (i.e., 584, 585, 586, 587, 588, 589 and 590),

[0295] 620-626 (i.e., 620, 621, 622, 623, 624, 625, 626),

[0296] 650-658 (i.e., 659, 651, 652, 653, 654, 655, 656, 657 and 658),

[0297] 665-668 (i.e., 666, 667 and 668),

[0298] 690-693 (i.e., 690, 691, 692 and 693),

[0299] 734-738 (i.e., 734, 735, 736, 737 and 738) and

[0300] 789-795 (i.e., 789, 790, 791, 792, 793, 794 and 795).

[0301] The following residues of Promozyme®are within a distance of 6 Åfrom the substrate and thus believed to be involved in interactions withsaid substrate:

[0302] 489, 551, 553, 555, 556, 620, 651, 691, 692, 791, 793, 794,506-510 (i.e., 507, 508, 509 and 510),

[0303] 586-588 (i.e., 586, 587 and 588),

[0304] 622-624 (i.e., 622, 623 and 624),

[0305] 653-656 (i.e., 653, 654, 655 and 656) and

[0306] 735-737 (i.e., 735, 736 and 737),

[0307] In a preferred embodiment of the invention, the parentpullulanase is modified in such a way that the variant pulluanaseexhibits an increased isoamylase activity compared to the parentpullulanase.

[0308] When used herein, the term “increased isoamylase activity” refersin general to the fact that the pullulanase variants according to theinvention exhibits a higher activity towards high molecular weightbranched starchy material, such as glycogen and amylopectin as comparedto the parent pullulanase, cf. above.

[0309] In an interesting embodiment of the invention the pullulanasevariant has an increased isoamylase activity as defined by an increaseof at least 5%, preferably of at least 10%, more preferably of at least15%, more preferably of at least 25%, most preferably of at least 50%,in particular of at least 75%, such as of at least 100% in the number ofreducing ends formed in the “assay for isoamylase-like activity”described herein, using 50 mM sodium acetate, a pH of 4.5, 5.0 or 5.5, atemperature of 60° C. and when incubated with a 10 w/v rabbit liverglycogen solution for a period of 10 min.

[0310] Similar modifications may be introduced in equivalent positionsof other pullulanases. Substitutions of particular interest are anycombination of one or both of the above with any of the othermodifications disclosed herein.

[0311] For example, the following residues of the pullulanase fromBacillus deramificans (SEQ ID NO: 3) are within a distance of 10 Å fromthe “docked” substrate and thus believed to be involved in interactionswith said substrate:

[0312] 435, 437, 485, 487, 488, 512, 677, 679, 682, 683, 729, 773, 784,492-494 (i.e., 492, 493 and 494),

[0313] 503-509 (i.e., 503, 504, 505, 506, 507, 508 and 509),

[0314] 549-557 (i.e., 549, 550, 551, 552, 553, 554, 555, 556 and 557),

[0315] 582-588 (i.e., 582, 583, 584, 585, 586, 587 and 588),

[0316] 618-624 (i.e., 618, 619, 620, 621, 622, 623 and 624),

[0317] 648-656 (i.e., 648, 649, 650, 651, 652, 653, 654, 655 and 656),

[0318] 663-666 (i.e., 663, 664, 665 and 666),

[0319] 688-691 (i.e., 688, 689, 690 and 691),

[0320] 732-736 (i.e., 732, 733, 734, 735 and 736) and

[0321] 787-793 (i.e., 787, 788, 879, 790, 791, 792 and 793).

[0322] The following residues of the pullulanase from Bacillusderamificans (SEQ ID NO: 3) are within a distance of 6 Å from thesubstrate and thus believed to be involved in interactions with saidsubstrate:

[0323] 487, 549, 551, 553, 554, 618, 649, 689, 690, 789, 791, 792,504-508 (i.e., 504, 505, 506, 507 and 508),

[0324] 584-586 (i.e., 584, 585 and 586),

[0325] 620-622 (i.e., 620, 621 and 622),

[0326] 651-654 (i.e., 651, 652, 653 and 654) and

[0327] 733-735 (i.e., 733, 734 and 735).

[0328] Examples of specific modifications in the above-mentioned regionsof Bacillus deramificans are: L621I/V, D508M/N/L/T/V, T586I/L/V,T677W/F/Y, Y729F/I/L, D679G/A/V, S732V/T/L/I, N735G/L/V/I/S/T/A and A(688-691).

[0329] Pullulanase Variants with Altered pH Dependent Activity Profile

[0330] The pH dependent activity profile can be changed by changing thepKa of residues within 15 Å, in particular by changing the pKa ofresidues within 10 Å, from the active site residues of the parentpullulanase. Changing the pKa of the active site residues is achieved,e.g., by changing the electrostatic interaction or hydrophobicinteraction between functional groups of amino acid side chains of agiven amino acid residue and its close surroundings. To obtain a higheractivity at a higher pH, negatively charged residues are placed near ahydrogen donor acid, whereas positively charged residues placed near anucleophilic acid will result in higher activity at low pH. Also, adecrease in the pKa can be obtained by reducing the accessibility ofwater or increasing hydrophobicity of the environment.

[0331] It is preferred that the variant in question exhibits a pHoptimum which is at least about 0.5 pH units higher or lower, preferablyat least about 1.0 pH units higher or lower, than the corresponding pHoptimum of the parent pullulanase when tested on a suitable substrate(e.g. pullulan, amylopectin or glycogen).

[0332] Furthermore, it is particular preferred that the variant inquestion exhibits an increased activity in the pH range of from 4 to 5.5as compared to the parent pullulanase when tested on a suitablesubstrate (e.g., pullulan, amylopectin or glycogen).

[0333] Thus, another aspect of the present invention relates to a methodfor constructing a variant of a parent pullulanase, the methodcomprising:

[0334] a) identifying an amino acid residue which is within 15 Å, inparticular within 10 Å, from an active site residue of the parentpullulanase in the three-dimensional structure of said parentpullulanse, and which is involved in electrostatic or hydrophobicinteractions with an active site residue;

[0335] b) substituting said amino acid residue with another amino acidresidue which changes the electrostatic and/or hydrophobic surroundingsof an active site residue, and which can be accommodated in thestructure;

[0336] c) optionally repeating steps a) and b) recursively;

[0337] d) optionally, making alterations each of which is an insertion,a deletion or a substitution of an amino acid residue at one or morepositions other than b);

[0338] e) preparing the variant resulting from steps a)-d);

[0339] f) testing the pH dependent activity of said variant; and

[0340] g) optionally repeating steps a)-f) recursively; and

[0341] h) selecting a variant having an altered pH dependent activity ascompared to the parent amylase.

[0342] In general, an amino acid residue which is within 15 Å or 10 Å,respectively, from an active site residue of the parent pullulanase maybe identified by using the INSIGHTII program.

[0343] In a preferred embodiment, the variant of a parent pullulanasehaving an altered pH dependent activity profile as compared to theparent pullulanase comprises a modification, e.g. a substitution, of anamino acid residue corresponding to one or more of the followingresidues of the amino acid sequence set forth in SEQ ID NO: 1 (allwithin 15A from the active site residues D736, E651, D622):

[0344] 430, 433, 518, 521, 565, 599, 600, 610, 611, 635, 636, 639, 717,760, 763, 764, 767, 817,

[0345] 435-443 (i.e., 435, 436, 437, 438, 439, 440, 441, 442, and 443),

[0346] 486-496 (i.e., 486, 487, 488, 489, 490, 491, 492, 493, 494, 495and 496),

[0347] 505-515 (i.e., 505, 506, 507, 508, 509, 510, 511, 512, 513, 514and 515),

[0348] 548-560 (i.e., 548, 549, 550, 551, 552, 553, 554, 555, 556, 557,558, 559 and 560),

[0349] 573-575, (i.e., 573, 574 and 575),

[0350] 583-595 (i.e., 583, 584, 585, 586, 587, 588, 589, 590, 591, 592,593, 594 and 594),

[0351] 602-604 (i.e., 602, 603 and 604),

[0352] 606-608 (i.e., 606-607 and 608),

[0353] 616-633 (i.e., 616, 617, 618, 619, 620, 621, 622, 623, 624, 625,626, 627, 628, 629, 630, 631, 632, and 633),

[0354] 646-672 (i.e., 646, 647, 648, 649, 650, 651, 652, 653, 654, 655,656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669,670, 671 and 672),

[0355] 674-696 (i.e., 674, 675, 676, 677, 678, 679, 680, 681, 682, 683,684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695 and 696),720-722 (i.e. 720, 721 and 722),

[0356] 725-747 (i.e., 725, 726, 727, 728, 729, 730, 731, 732, 733, 734,735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746 and 747),773-781 (i.e. 773, 774, 775, 776, 777, 778, 779, 780 and 781), 783-797(i.e. 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795,796 and 797) and

[0357] 799-802 (i.e., 799, 800, 801 and 802).

[0358] Within 10 Å from the active site residues D736, E651, D622:

[0359] 437, 442, 492, 514, 575, 594, 603, 632, 635, 684, 688, 691, 692,721, 727, 729, 742, 743, 775, 777, 778, 780, 784, 786, 800, 487-490(i.e., 487, 488, 489 and 490),

[0360] 507-511 (i.e., 507, 508, 509, 510 and 511),

[0361] 550-557 (i.e., 550, 551, 552, 553, 554, 555, 556 and 556),

[0362] 585-588 (i.e., 585, 586, 587 and 588),

[0363] 590-592 (i.e., 590, 591 and 592),

[0364] 619-628 (i.e., 619, 620, 621, 622, 623, 624, 625, 626, 627 and

[0365] 628), 648-655 (i.e., 648, 649, 650, 651, 652, 653, 654 and 655),

[0366] 665-671 (i.e., 665, 666, 667, 668, 669, 670 and 671),

[0367] 676-681 (i.e., 676, 677, 678, 679, 680 and 681),

[0368] 731-740 (i.e., 731, 732, 733, 734, 735, 736, 737, 738, 739 and740) and

[0369] 788-793 (i.e., 788, 789, 790, 791, 792 and 793).

[0370] Similar modifications may be introduced in equivalent positionsof other pullulanases. Variants of particular interest have acombination of one or more of the above with any of the othermodifications disclosed herein.

[0371] Thus, in another preferred embodiment, the variant of a parentpullulanase having an altered pH dependent activity profile as comparedto the parent pullulanase comprises a modification, e.g., asubstitution, of an amino acid residue corresponding to one or more ofthe following residues of the amino acid sequence set forth in SEQ IDNO: 3 (all within 15 Å from the active site residues D734, E649 andD620):

[0372] 428, 431, 516, 519, 563, 597, 598, 608, 609, 633, 634, 637, 715,758, 761, 762, 765, 815, 433-441 (i.e., 433, 434, 435, 436, 437, 438,439, 440 and 441),

[0373] 484-494 (i.e., 484, 485, 486, 487, 488, 489, 490, 491, 492, 493and 494),

[0374] 503-513 (i.e., 503, 504, 505, 506, 507, 508, 509, 510, 511, 512and 513),

[0375] 546-558 (546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556,557 and 558),

[0376] 571-573 (i.e., 571, 572 and 573),

[0377] 581-593 (i.e., 581, 582, 583, 584, 585, 586, 587, 588, 589, 590,591, 592 and 593),

[0378] 600-602 (i.e., 600, 601 and 602),

[0379] 604-606 (i.e., 604, 605 and 606),

[0380] 614-631 (i.e., 614, 615, 616, 617, 618, 619, 620, 621, 622, 623,624, 625, 626, 627, 628, 629, 630 and 631),

[0381] 644-670 (i.e., 644, 645, 646, 647, 648, 649, 650, 651, 652, 653,654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667,668, 669 and 670),

[0382] 672-694 (i.e., 672, 673, 674, 675, 676, 677, 678, 679, 680, 681,682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693 and 694),

[0383] 718-720 (i.e., 718, 719 and 720), 723-745 (i.e., 723, 734, 725,726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739,740, 741, 742, 743, 744 and 745),

[0384] 771-779 (i.e., 771, 772, 773, 774, 775, 776, 777, 778 and 779),781-795 (i.e. 781, 782., 783, 784, 785, 786, 787, 788, 789, 790, 791,792, 793, 794 and 795) and

[0385] 797-800 (i.e., 797, 798, 799 and 800).

[0386] Within 10 Å from the active site residues D734, E649 and D620:

[0387] 435, 440, 490, 512, 573, 601, 605, 630, 669, 682, 686, 689, 690,719, 725, 727, 740, 741, 773, 775, 776, 778, 782, 784, 798, 485-488(i.e., 485, 486, 487 and 488),

[0388] 505-509 (i.e., 505, 506, 507, 508 and 509),

[0389] 548-555 (i.e., 548, 549, 550, 551, 552, 553, 554 and 555),

[0390] 583-586 (i.e., 583, 584, 585 and 586),

[0391] 588-590 (i.e., 588, 589 and 590),

[0392] 617-626 (i.e., 616, 617, 618, 619, 620, 621, 622, 623, 624, 625and 626),

[0393] 646-653 (i.e., 646, 647, 648, 649, 650, 651, 652 and 653),

[0394] 663-667 (i.e., 663, 664, 665, 666 and 667),

[0395] 674-679 (i.e., 674, 675, 676, 677, 678 and 679),

[0396] 729-738 (i.e., 729, 730, 731, 732, 733, 734, 735, 736, 737 and738) and

[0397] 786-791 (i.e., 786, 787, 788, 789, 790 and 791).

[0398] Specific examples of substitutions in the above-mentionedpositions include D437L/I/V/F, D440L/I/V/F, M486K, M487K, D503L/I/V/F,D508N/L/T/V, T586V/I, M630H and D437L/I/V/F+D440L/I/V/F+D503L/I/V/F.

[0399] Nomenclature for Amino Acid Modifications

[0400] The nomenclature used herein for defining modifications isessentially as described in WO 92/05249. Thus, G406A indicates asubstitution of the amino acid G (Gly) in position 406 with the aminoacid A (Ala). G406 indicates a substitution of the amino acid G (Gly)with any other amino acid. P394F/W/I/L or P394F, W, I, L indicates asubstitution of P394 with F, W, I or L. Δ(688-691) indicates a deletionof amino acids in positions 688-691. 412-A-413 indicates an insertion ofA between amino acids 412 and 413.

[0401] A deletion of Alanine in position A30 is shown as:

[0402] A30*

[0403] and insertion of an additional amino acid residue, such asLysine, is shown as:

[0404] A30AK

[0405] Where a specific pullulanase contains a “deletion” in comparisonwith other pullulanases and an insertion is made in such a position thisis indicated as:

[0406] *36D

[0407] for insertion of an Aspartic acid in position 36.

[0408] Multiple mutations are separated by plus signs, i.e.:

[0409] A30N+E34S or A30N/E34S

[0410] representing mutations in positions 30 and 34 substitutingAlanine and Glutamic acid for Asparagine and Serine, respectively.

[0411] Furthermore, when a position suitable for modification isidentified herein without any specific modification being suggested, itis to be understood that any amino acid residue may be substituted forthe amino acid residue present in the position. Thus, for instance, whena modification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the Alanine may be deleted orsubstituted for any other amino acid, i.e., any one of: R, N, D, A, C,Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

[0412] Further, “A30X” means any one of the following substitutions:A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K, A30M,A30F, A30P, A30S, A30T, A30W, A30Y, or A30 V; or in short: A30R, N, D,C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

[0413] When used herein, the term “modification” (of a particular aminoacid residue) is intended to cover substitution and deletion (of theparticular amino acid residue) as well as insertion of one or more aminoacid residues after the particular amino acid residue.

[0414] Polypeptide Sequence Homology

[0415] For purposes of the present invention, the degree of homology maybe suitably determined according to the method described in S. B.Needleman and C. D. Wunsch, Journal of Molecular Biology, 48, 443-45,with the following settings for polypeptide sequence comparison: GAPcreation penalty of 3.0 and GAP extension penalty of 0.1. Thedetermination may be done by means of a computer program known such asGAP provided in the UWGCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711).

[0416] Hybridization

[0417] Suitable experimental conditions for determining hybridizationbetween a nucleotide probe and a homologous DNA or RNA sequence involvespresoaking of the filter containing the DNA fragments or RNA tohybridize in 5×SSC (sodium chloride/sodium citrate, Sambrook, et al.Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor,1989) for 10 min, and prehybridization of the filter in a solution of5×SSC, 5× Denhardt's solution (Sambrook, et al., 1989), 0.5% SDS and 100micro g/ml of denatured sonicated salmon sperm DNA (Sambrook, et al.,1989), followed by hybridization in the same solution containing arandom-primed (A. P. Feinberg B. and Vogelstein, Anal. Biochem. 132,6-13 (1983)), ³²P-dCTP-labeled (specific activity>1×10⁹ cpm/micro g)probe for 12 hours at ca. 45° C. The filter is then washed twice for 30minutes in 2×SSC, 0.5% SDS at least 55° C. (low stringency), preferablyat least 60° C. (medium stringency), more preferably at least 65° C.(medium/high stringency), more preferably at least 70° C. (highstringency), even more preferably at least 75° C. (very highstringency).

[0418] Molecules which hybridize to the oligonucleotide probe underthese conditions are detected by exposure to x-ray film.

[0419] Methods of Preparing Pullulanase Variants According to theInvention

[0420] Cloning a DNA Sequence Encoding a Pullulanase

[0421] The DNA sequence encoding a parent pullulanase may be isolatedfrom any cell or microorganism producing the pullulanase in question,using various methods well known in the art.

[0422] First, a genomic DNA and/or cDNA library should be constructedusing chromosomal DNA or messenger RNA from the organism that producesthe pullulanase to be studied. Then, if the amino acid sequence of thepullulanase is known, homologous, labelled oligonucleotide probes may besynthesised and used to identify pullulanase-encoding clones from agenomic library prepared from the organism in question. Alternatively, alabelled oligonucleotide probe containing sequences homologous to aknown pullulanase gene could be used as a probe to identifypullulanase-encoding clones, using hybridization and washing conditionsof lower stringency.

[0423] Alternatively, the DNA sequence encoding the enzyme may beprepared synthetically by established standard methods, e.g. thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers,Tetrahedron Letters, 22, 1859-1869 (1981) or the method described byMatthes et al. The EMBO, 3, 801-805 (1984). In the phosphoroamiditemethod, oligonucleotides are synthesized, e.g., in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors.

[0424] Finally, the DNA sequence may be of mixed genomic and syntheticorigin, mixed synthetic and cDNA origin or mixed genomic and cDNAorigin, prepared by ligating fragments of synthetic, genomic or cDNAorigin, wherein the fragments correspond to various parts of the entireDNA sequence, in accordance with techniques well known in the art. TheDNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or R. K. Saiki et al. Science, 239, 487-491(1988).

[0425] Site-Directed Mutagenesis

[0426] Once a pullulanase-encoding DNA sequence has been isolated, anddesirable sites for modification identified, modifications may beintroduced using synthetic oligonucleotides. These oligonucleotidescontain nucleotide sequences flanking the desired modification sites;mutant nucleotides are inserted during oligonucleotide synthesis. In aspecific method, a single-stranded gap of DNA, bridging thepullulanase-encoding sequence, is created in a vector carrying thepullulanase gene. Then the synthetic nucleotide, bearing the desiredmodification, is annealed to a homologous portion of the single-strandedDNA. The remaining gap is then filled in with DNA polymerase I (Klenowfragment) and the construct is ligated using T4 ligase. A specificexample of this method is described in Morinaga et al. Biotechnology 2,639-646 (1984). U.S. Pat. No. 4,760,025 disclose the introduction ofoligonucleotides encoding multiple modifications by performing minoralterations of the cassette. However, an even greater variety ofmodifications can be introduced at any one time by the Morinaga methodbecause a multitude of oligonucleotides, of various lengths, can beintroduced.

[0427] Another method of introducing modifications into apullulanase-encoding DNA sequences is described in Nelson and LongAnalytical Biochemistry, 180, 147-151 (1989). It involves a 3-stepgeneration of a PCR fragment containing the desired modificationintroduced by using a chemically synthesized DNA strand as one of theprimers in the PCR reactions. From the PCR-generated fragment, a DNAfragment carrying the modification may be isolated by cleavage withrestriction endonucleases and reinserted into an expression plasmid.

[0428] Random Mutagenesis

[0429] Random mutagenesis is suitably performed either as localized orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

[0430] The random mutagenesis of a DNA sequence encoding a parentpullulanase may be conveniently performed by use of any method known inthe art.

[0431] In relation to the above, a further aspect of the presentinvention relates to a method for generating a variant of a parentpullulanase, wherein the variant exhibits an altered property, such asincreased thermostability, increased stability at low pH and at lowcalcium concentration, relative to the parent pullulanase, the methodcomprising:

[0432] (a) subjecting a DNA sequence encoding the parent pullulanase torandom mutagenesis,

[0433] (b) expressing the mutated DNA sequence obtained in step (a) in ahost cell, and

[0434] (c) screening for host cells expressing a pullulanase variantwhich has an altered property relative to the parent pullulanase.

[0435] Step (a) of the above method of the invention is preferablyperformed using doped primers.

[0436] For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents. The mutagenizing agentmay, e.g., be one which induces transitions, transversions, inversions,scrambling, deletions, and/or insertions.

[0437] Examples of a physical or chemical mutagenizing agent suitablefor the present purpose include ultraviolet (UV) irradiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogues. When such agents areused, the mutagenesis is typically performed by incubating the DNAsequence encoding the parent enzyme to be mutagenized in the presence ofthe mutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

[0438] When the mutagenesis is performed by the use of anoligonucleotide, the oligonucleotide may be doped or spiked with thethree non-parent nucleotides during the synthesis of the oligonucleotideat the positions that are to be changed. The doping or spiking may bedone so that codons for unwanted amino acids are avoided. The doped orspiked oligonucleotide can be incorporated into the DNA encoding thepullulaase enzyme by any published technique, using, e.g., PCR, LCR orany DNA polymerase and ligase as deemed appropriate.

[0439] Preferably, the doping is carried out using “constant randomdoping”, in which the percentage of wild-type and modification in eachposition is predefined. Furthermore, the doping may be directed toward apreference for the introduction of certain nucleotides, and thereby apreference for the introduction of one or more specific amino acidresidues. The doping may be made, e.g., so as to allow for theintroduction of 90% wild type and 10% modifications in each position. Anadditional consideration in the choice of a doping scheme is based ongenetic as well as protein-structural constraints. The doping scheme maybe made by using the DOPE program which, inter alia, ensures thatintroduction of stop codons is avoided (L. J. Jensen et al. Nucleic AcidResearch, 26, 697-702 (1998).

[0440] When PCR-generated mutagenesis is used, either a chemicallytreated or non-treated gene encoding a parent pullulanase enzyme issubjected to PCR under conditions that increase the misincorporation ofnucleotides (Deshler 1992; Leung et al., Technique, 1, 1989, pp. 11-15).

[0441] A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet.,133, 1974, 179-191), S. cereviseae or any other microbial organism maybe used for the random mutagenesis of the DNA encoding the pullulanaseby, e.g., transforming a plasmid containing the parent enzyme into themutator strain, growing the mutator strain with the plasmid andisolating the mutated plasmid from the mutator strain. The mutatedplasmid may be subsequently transformed into the expression organism.

[0442] The DNA sequence to be mutagenized may conveniently be present ina genomic or cDNA library prepared from an organism expressing theparent pullulanase. Alternatively, the DNA sequence may be present on asuitable vector such as a plasmid or a bacteriophage, which as such maybe incubated with or otherwise exposed to the mutagenising agent. TheDNA to be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharbored in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

[0443] In some cases it may be convenient to amplify the mutated DNAsequence prior to performing the expression step b) or the screeningstep c). Such amplification may be performed in accordance with methodsknown in the art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme.

[0444] Subsequent to the incubation with or exposure to the mutagenisingagent, the mutated DNA is expressed by culturing a suitable host cellcarrying the DNA sequence under conditions allowing expression to takeplace. The host cell used for this purpose may be one which has beentransformed with the mutated DNA sequence, optionally present on avector, or one which was carried the DNA sequence encoding the parentenzyme during the mutagenesis treatment. Examples of suitable host cellsare the following: gram positive bacteria such as Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram negative bacteria such as E. coli.

[0445] The mutated DNA sequence may further comprise a DNA sequenceencoding functions permitting expression of the mutated DNA sequence.

[0446] Localized Random Mutagenesis

[0447] The random mutagenesis may be advantageously localized to a partof the parent pullulanase in question. This may, e.g., be advantageouswhen certain regions of the enzyme have been identified to be ofparticular importance for a given property of the enzyme, and whenmodified are expected to result in a variant having improved properties.Such regions may normally be identified when the tertiary structure ofthe parent enzyme has been elucidated and related to the function of theenzyme.

[0448] The localized, or region-specific, random mutagenesis isconveniently performed by use of PCR generated mutagenesis techniques asdescribed above or any other suitable technique known in the art.Alternatively, the DNA sequence encoding the part of the DNA sequence tobe modified may be isolated, e.g., by insertion into a suitable vector,and said part may be subsequently subjected to mutagenesis by use of anyof the mutagenesis methods discussed above.

[0449] General Method for Random Mutagenesis by use of the DOPE Program

[0450] The random mutagenesis may be carried out by the following steps:

[0451] 1. Select regions of interest for modification in the parentenzyme

[0452] 2. Decide on mutation sites and non-mutated sites in the selectedregion

[0453] 3. Decide on which kind of mutations should be carried out, e.g.with respect to the desired stability and/or performance of the variantto be constructed

[0454] 4. Select structurally reasonable mutations

[0455] 5. Adjust the residues selected by step 3 with regard to step 4.

[0456] 6. Analyze by use of a suitable dope algorithm the nucleotidedistribution.

[0457] 7. If necessary, adjust the wanted residues to genetic coderealism, e.g. taking into account constraints resulting from the geneticcode, e.g. in order to avoid introduction of stop codons; the skilledperson will be aware that some codon combinations cannot be used inpractice and will need to be adapted

[0458] 8. Make primers

[0459] 9. Perform random mutagenesis by use of the primers

[0460] 10. Select resulting pullulanase variants by screening for thedesired improved properties.

[0461] Suitable dope algorithms for use in step 6 are well known in theart. One such algorithm is described by Tomandl, D. et al., 1997,Journal of Computer-Aided Molecular Design 11:29-38. Another algorithmis DOPE (Jensen, L J, Andersen, K V, Svendsen, A, and Kretzschmar, T(1998) Nucleic Acids Research 26:697-702).

[0462] Expression of Pullulanase Variants

[0463] The construction of the variant of interest is accomplished bycultivating a microorganism comprising a DNA sequence encoding thevariant under conditions which are conducive for producing the variant,and optionally subsequently recovering the variant from the resultingculture broth. This is described in detail further below.

[0464] According to the invention, a DNA sequence encoding the variantproduced by methods described above, or by any alternative methods knownin the art, can be expressed, in the form of a protein or polypeptide,using an expression vector which typically includes control sequencesencoding a promoter, operator, ribosome binding site, translationinitiation signal, and, optionally, a repressor gene or variousactivator genes.

[0465] The recombinant expression vector carrying the DNA sequenceencoding an pullulanase variant of the invention may be any vector whichmay conveniently be subjected to recombinant DNA procedures, and thechoice of vector will often depend on the host cell into which it is tobe introduced. Thus, the vector may be an autonomously replicatingvector, i.e., a vector that exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

[0466] In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence thatshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding a pullulanase variant of theinvention, especially in a bacterial host, are the promoter of the lacoperon of E. coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis α-amylase gene(amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amyloliquefaciensalpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA andxylB genes, etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralalpha-amylase, A. niger acid stable alpha-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase (TPI) or A. nidulans acetamidase.

[0467] The expression vector of the invention may also comprise asuitable transcription terminator and, in eukaryotes, polyadenylationsequences operably connected to the DNA sequence encoding thepullulanase variant of the invention. Termination and polyadenylationsequences may suitably be derived from the same sources as the promoter.

[0468] The vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and pIJ702.

[0469] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance. Furthermore, the vector may compriseAspergillus selection markers such as amds, argB, niaD and sC, a markergiving rise to hygromycin resistance, or the selection may beaccomplished by co-transformation, e.g., as described in WO 91/17243.

[0470] While intracellular expression may be advantageous in somerespects, e.g., when using certain bacteria as host cells, it isgenerally preferred that the expression is extracellular. In general,the Bacillus α-amylases mentioned herein comprise a preregion permittingsecretion of the expressed protease into the culture medium. Ifdesirable, this preregion may be replaced by a different preregion orsignal sequence, conveniently accomplished by substitution of the DNAsequences encoding the respective preregions.

[0471] The procedures used to ligate the DNA construct of the inventionencoding the pullulanase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al. MolecularCloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor, 1989).

[0472] The cell of the invention, either comprising a DNA construct oran expression vector of the invention as defined above, isadvantageously used as a host cell in the recombinant production of apullulanase variant of the invention. The cell may be transformed withthe DNA construct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g. by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

[0473] The cell of the invention may be a cell of a higher organism suchas a mammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

[0474] Examples of suitable bacteria are gram positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram negative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

[0475] The yeast organism may favourably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g. Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

[0476] In a yet further aspect, the present invention relates to amethod for producing a pullulanase variant of the invention, the methodcomprising: cultivating a host cell as described above under conditionsconducive to the production of the variant and recovering the variantfrom the cells and/or culture medium.

[0477] The medium used to cultivate the cells may be any conventionalmedium suitable for growing the host cell in question and obtainingexpression of the pullulanase variant of the invention. Suitable mediaare available from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

[0478] The pullulanase variant secreted from the host cells mayconveniently be recovered from the culture medium by well-knownprocedures, including separating the cells from the medium bycentrifugation or filtration, and precipitating proteinaceous componentsof the medium by means of a salt such as ammonium sulfate, followed bythe use of chromatographic procedures such as ion exchangechromatography, affinity chromatography, or the like.

[0479] Testing of Pullulanase

[0480] Pullulanase variants produced by any of the methods describedabove may be tested, either prior to or after purification, forpullulanase activity in a screening assay which measures the ability ofthe variant to degrade pullulan or, in case it is desired to screen foran increased isoamylases activity, the ability of the variant to degradeamylopectin. The screening in step 10 in the above-mentioned randommutagenesis method of the invention may be conveniently performed by useof a filter assay based on the following procedure: A microorganismcapable of expressing the mutated pullulanase of interest is incubatedon a suitable medium and under suitable conditions for secretion of theenzyme, the medium being covered with two filters comprising aprotein-binding filter placed under a second filter exhibiting a lowprotein binding capability. The microorganism is grown on the second,top filter. Subsequent to the incubation, the bottom protein-bindingfilter comprising enzymes secreted from the microorganism is separatedfrom the second filter comprising the microorganism. The protein-bindingfilter is then subjected to screening for the desired enzymaticactivity, and the corresponding microbial colonies present on the secondfilter are identified. The first filter used for binding the enzymaticactivity may be any protein-binding filter, e.g., nylon ornitrocellulose. The second filter carrying the colonies of theexpression organism may be any filter that has no or low affinity forbinding proteins, e.g., cellulose acetate or DURAPORA™.

[0481] Screening consists of treating the first filter to which thesecreted protein is bound with a substrate that allows detection of theactivity. The enzymatic activity may be detected by a dye, fluorescence,precipitation, pH indicator, IR-absorbance or any other known techniquefor detection of enzymatic activity. The detecting compound may beimmobilized by any immobilizing agent, e.g., agarose, agar, gelatine,polyacrylamide, starch, filter paper, cloth; or any combination ofimmobilizing agents. For example, isoamylase activity can be detected byCibacron Red labelled amylopectin, which is immobilized in agarose.isoamylase activity on this substrate produces zones on the plate withreduced red color intensity (clearing zones).

[0482] To screen for variants with increased stability, the filter withbound pullulanase variants can be pretreated prior to the detection stepdescribed above to inactivate variants that do not have improvedstability relative to the parent pullulanase. This inactivation step mayconsist of, but is not limited to, incubation at elevated temperaturesin the presence of a buffered solution at any pH from pH 2 to 12, and/orin a buffer containing another compound known or thought to contributeto altered stability, e.g., surfactants, EDTA, EGTA, wheat flourcomponents, or any other relevant additives. Filters so treated for aspecified time are then rinsed briefly in deionized water and placed onplates for activity detection as described above. The conditions arechosen such that stabilized variants show increased enzymatic activityrelative to the parent after incubation on the detection media.

[0483] To screen for variants with altered thermostability, filters withbound variants are incubated in buffer at a given pH (e.g., in the rangefrom pH 2-12) at an elevated temperature (e.g., in the range from50°-110° C.) for a time period (e.g., from 1-20 minutes) to inactivatenearly all of the parent pullulanase, rinsed in water, then placeddirectly on a detection plate containing immobilized Cibacron Bluelabeled pullulan and incubated until activity is detectable. As will beunderstood, thermostability and increased isoamylase activity may betested simultaneously by using a detection plate containing immobilizedCibacron Red labeled amylopectin and incubate until activity isdetectable. Moreover, pH dependent stability can be screened for byadjusting the pH of the buffer in the above inactivation step such thatthe parent pullulanase is inactivated, thereby allowing detection ofonly those variants with increased stability at the pH in question. Toscreen for variants with increased calcium-dependent stability, calciumchelators, such as ethylene glycol-bis(beta-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), is added to the inactivation bufferat a concentration such that the parent pullulanase is inactivated underconditions further defined, such as buffer pH, temperature or aspecified length of incubation.

[0484] The variants of the invention may be suitably tested by assayingthe pullulan- or amylopectin-degrading activity of the variant, forinstance by growing host cells transformed with a DNA sequence encodinga variant on a starch-containing agarose plate and identifying pullulan-and/or amylopectin-degrading host cells as described above. Furthertesting in regard to altered properties, including specific activity,substrate specificity, cleavage pattern, thermoactivation,thermostability, pH dependent activity or optimum, pH dependentstability, temperature dependent activity or optimum, transglycosylationactivity, stability, and any other parameter of interest, may beperformed on purified variants in accordance with methods known in theart as described below.

[0485] Finally the present invention relates to the used of apullulanase variant of the invention for starch conversion, both for theliquefaction and saccharification steps, in particular for producingsyrups, such as dextrose or maltose syrups. A pullulanase variant of theinvention may also be used for producing sweeteners; ethanol, such asfuel, drinking and industrial ethanol, from starch or whole grains (seefor instance U.S. Pat. No. 5,231,017-A or U.S. Pat. No. 5,756,714-Ahereby incorporated by reference). Further, a pullulanase variant of theinvention may also be used as cleaning ingredient, in laundry detergentcompositions, dishwashing detergent, and hard surface cleaningcompositions (see e.g., WO 99/23211, WO 97/07202 or WO 96/238874 fordetails on examples on cleaning compositions ingredients, the referenceshereby being incorporated by reference). Normally a cleaning ordetergent composition also comprises at least a protease, in particularBacillus proteases, and also one or more of the following activities:alpha-amylase, lipase, cellulase, mannanase, CGTase, maltogenic amylase.

[0486] The invention is further illustrated with reference to thefollowing examples that are not intended to be in any way limiting tothe scope of the invention as claimed.

[0487] Determination of Pullulanase Activity

[0488] Endo-pullulanase activity in NPUN is measured relative to aNovozymes pullulanase standard. One pullulanase unit (NPUN) is definedas the amount of enzyme that releases 1 micro mol glucose per minuteunder the standard conditions (0.7% red pullulan (Megazyme), pH 5, 40°C., 20 minutes). The activity is measured in NPUN/ml using red pullulan.1 ml diluted sample or standard is incubated at 40° C. for 2 minutes.0.5 ml 2% red pullulan, 0.5 M KCl, 50 mM citric acid, pH 5 are added andmixed. The tubes are incubated at 40° C. for 20 minutes and stopped byadding 2.5 ml 80% ethanol. The tubes are left standing at roomtemperature for 10-60 minutes followed by centrifugation 10 minutes at4000 rpm. OD of the supernatants is then measured at 510 nm and theactivity calculated using a standard curve.

[0489] Expression of Pullulanase from Bacillus deramificans

[0490] The pullulanase from Bacillus deramificans (SEQ ID NO: 3) isexpressed in B. subtilis from a plasmid denoted pCA36. This plasmidcontains the complete gene encoding the pullulanase, the expression ofwhich is directed by the promoter from Bacillus amyloliquefaciensalpha-amylase. Further, the plasmid contains the origin of replication,oriT, from plasmid pUB110 and the cat gene from plasmid pC194 conferringresistance towards chloramphenicol. PCA36 is shown in FIG. 1.

EXAMPLES Example 1

[0491] Construction of Bacillus deramificans D620A Variant

[0492] Gene specific primer 132011 and mutagenic primer 132012 are usedto amplify by PCR an approximately 410 bp DNA fragment from the pCA36plasmid.

[0493] The 410 bp fragment is purified from an agarose gel and used as aMega-primer together with primer 136054 in a second PCR carried out onthe same template.

[0494] The resulting approximately 1110 bp fragment is digested withrestriction enzymes BsiW I and Mlu I and the resulting approximately 330bp DNA fragment is purified and ligated with the pCA36 plasmid digestedwith the same enzymes. Competent Bacillus subtilis SHA273 (amylase andprotease low) cells are transformed with the ligation and chlorampenicolresistant transformants are checked by colony PCR.

[0495] The mutagenesis primer 132012 introduced the D620A substitution(written in bold in the primer sequence) and introduced simultaneously aBgl I restriction site (underlined in the primer seq.), whichfacilitates easy pinpointing of mutants.

[0496] Finally, DNA sequencing was carried out to verify the presence ofthe correct mutations on the plasmid. Primer 132011:5′ cgcttcggaatcattaggattgc 3′ (SEQ ID NO:7) Primer 132012:5′ gcttccgttttgccttaatggcgctgc 3′ (SEQ ID NO:8) Primer 136054:5′ ggccaaggctctacccgaacggc 3′ (SEQ ID NO:9)

Example 2

[0497] Construction of Bacillus deramificans E649A Variant

[0498] This variant constructed as described in Example 1, except thatmutagenic primer 132013 is used. The mutagenesis primer 132013introduced the E649A substitution (written in bold in the primersequence) and a NarI restriction site (underlined in the primersequence), which facilitates easy pinpointing of mutants.

[0499] Primer 132013:

[0500] 5′ gcactttacggggcgccatggacggg 3′ (SEQ ID NO: 10)

Example 3

[0501] Thermostability test of variant of the invention The belowmentioned variants were constructed in Bacillus deramificanspullullanase using a megaprimer approach similar to the one describedabove. The following primers were used in order to introduce the variousamino acid changes indicated below (all primer are written directional(5′-3′): deletion (1-111) Nr.167 TVB400:CATTCTGCAGCGGCCGCAAACGCTTATTTAGATGCTTCAAACC (SEQ ID NO:11) deletion(1-113) Nr.168-tvb401: CATTCTGCAGCGGCCGCAGATGATCTTGGGAATACCTATAC (SEQ IDNO:12) D562P Nr.170-tvb496: CTTTGCCACGCAGATCTCTCCCTTCGATAAAATTG (SEQ IDNO:13) G292P NR.171-tvb497: CATTCAAACGGATCCCTATCAGGCAAAG (SEQ ID NO:14)G794P Nr.172-tvb498: GTTATAATGCACCCGATGCGGTCAATG (SEQ ID NO:15) D148PNr. 173-tvb499: CAGCAAATAAGCCCATTCCAGTGACATCTGTG (SEQ ID NO:16) N119PNr. 174-tvb500: CTTATTTAGATGCATCACCCCAGGTGC (SEQ ID NO:17) N400SNr.175-tvb567: CAACTGCGATCGCACCAAGTGGAACGAG (SEQ ID NO:18) N400LNr.176-tvb568: GCGATCGCACCACTTCGAACGAGAGGC (SEQ ID NO:19) N400FNr.177-tvb569: CTGCGATCGCACCATTTGGAACGAGAGGC (SEQ ID NO:20) N446SNr.178-tvb570: GACTTTTCAATTGACCCTTCTTCGGGTAT (SEQ ID NO:21) N446LNr.179-tvb571: GTCCGTGACTTTTCAATTGACCCTCTTTCGGG (SEQ ID NO:22) N446FNr.180-tvb572: GTCCGTGACTTTTCAATTGACCCTTTTTCGGGTATG (SEQ ID NO:23) N504SNr.181-tvb573: CCAAGATAGTTGGGGTTACGATCCTCGCAAC (SEQ ID NO:24) N504LNr.182-tvb574: CCAAGATCTTTGGGGTTACGATCCTCGC (SEQ ID NO:25) N504FNr.183-tvb575: CCCAAGATTTTTGGGGTTACGATCCTCGC (SEQ ID NO:26) N735SNr.184-tvb576: GTCACAAGTCACGATAGCTACACCCTTTGGG (SEQ ID NO:27) N735L Nr.185-tvb577: GTCACAAGTCACGATCTCTACACCCTTTGGGAC (SEQ ID NO:28) N735F Nr.186-tvb578: GTCACAAGTCACGATTTCTACACCCTTTGGG (SEQ ID NO:29) N789SNr.187-tvb579: GCAACGACAGTAGTTATAATGCCGGCGATG (SEQ ID NO:30) N789LNr.188-tvb580 GCAACGACCTTAGTTATAATGCCGGCGATG (SEQ ID NO:31) N789FNr.189-tvb581 GCAACGACTTTAGTTATAATGCCGGCGATG (SEQ ID NO:32) I566ANr.190-TVB582 GACTTCGATAAAGCGGTACCAGAATATTATTACC (SEQ ID NO:33) Q485HNr. 191 TVB 583 GGGATTACACATGTTCATCTTATGCCTGTTTTCG (SEQ ID NO:34) V551INr. 192 TVB 584 CATTGGGGTCAACATGGATGTTATCTATAATCATACC (SEQ ID NO:35)S492F Nr. 193 TVB 585 GTTTTCGCATTTAACAGTGTCGACGAAACTGATCC (SEQ ID NO:36)D444R Nr. 194 TVB 586 GACTTTTCCATTCGCCCGAATTCGGGTATG (SEQ ID NO:37)D444K Nr 195 TVB 587 CGTGACTTTTCCATTAAACCGAATTCGGGTATG (SEQ ID NO:38)

[0502] The deletion of an internal fragment corresponding to amino acids154 to amino acid 273 was done by SOE PCR (Horton et al, 1989, Gene 77:pp61-68) utilizing the following two overlap generatingoligonucleotides: 1) CCCTAGAGTAACAGATGTCACTGGAATATCC (SEQ ID NO:39) 2)GGATATTCCAGTGACATCTGTTACTCTAGGGG (SEQ ID NO:40)

[0503]Bacillus subtilis SHA273 was transformed with plasmids harbouringthe various variants and fermented.

[0504] The fermentation supernatant containing pullulanase variant issubjected to the stability assay (Thermostability Assay 2 (T½)) in orderto determine T½ values of inactivation using the assay described above.

1 40 1 2766 DNA Bacillus acidopullulyticus CDS (1)..(2766) 1 gat tct acttcg act aaa gtt att gtt cat tat cat cgt ttt gat tcc 48 Asp Ser Thr SerThr Lys Val Ile Val His Tyr His Arg Phe Asp Ser 1 5 10 15 aac tat acgaat tgg gac gtc tgg atg tgg cct tat cag cct gtt aat 96 Asn Tyr Thr AsnTrp Asp Val Trp Met Trp Pro Tyr Gln Pro Val Asn 20 25 30 ggt aat gga gcagct tac caa ttc act ggt aca aat gat gat ttt ggc 144 Gly Asn Gly Ala AlaTyr Gln Phe Thr Gly Thr Asn Asp Asp Phe Gly 35 40 45 gct gtt gca gat acgcaa gtg cct gga gat aat aca caa gtt ggt ttg 192 Ala Val Ala Asp Thr GlnVal Pro Gly Asp Asn Thr Gln Val Gly Leu 50 55 60 att gtt cgt aaa aat gattgg agc gag aaa aat aca cca aac gat ctc 240 Ile Val Arg Lys Asn Asp TrpSer Glu Lys Asn Thr Pro Asn Asp Leu 65 70 75 80 cat att gac ctt gca aaaggc cat gaa gta tgg att gta caa ggg gat 288 His Ile Asp Leu Ala Lys GlyHis Glu Val Trp Ile Val Gln Gly Asp 85 90 95 cca act att tat tac aat ctgagc gac gca cag gct gcc gca ata cca 336 Pro Thr Ile Tyr Tyr Asn Leu SerAsp Ala Gln Ala Ala Ala Ile Pro 100 105 110 tct gtt tca aat gcc tat cttgat gat gaa aaa aca gta cta gca aag 384 Ser Val Ser Asn Ala Tyr Leu AspAsp Glu Lys Thr Val Leu Ala Lys 115 120 125 cta agt atg ccg atg acg ctggcg gat gct gca agc ggc ttt acg gtt 432 Leu Ser Met Pro Met Thr Leu AlaAsp Ala Ala Ser Gly Phe Thr Val 130 135 140 ata gat aaa acc aca ggt gaaaaa atc cct gtc acc tct gct gta tcc 480 Ile Asp Lys Thr Thr Gly Glu LysIle Pro Val Thr Ser Ala Val Ser 145 150 155 160 gca aat ccg gta act gccgtt ctt gtt gga gat tta caa cag gct ttg 528 Ala Asn Pro Val Thr Ala ValLeu Val Gly Asp Leu Gln Gln Ala Leu 165 170 175 gga gca gcg aat aat tggtca cca gat gat gat cac aca ctg cta aaa 576 Gly Ala Ala Asn Asn Trp SerPro Asp Asp Asp His Thr Leu Leu Lys 180 185 190 aag ata aat cca aac ctttac caa tta tcg ggg aca ctt cca gct ggt 624 Lys Ile Asn Pro Asn Leu TyrGln Leu Ser Gly Thr Leu Pro Ala Gly 195 200 205 aca tac caa tat aag atagcc ttg gac cat tct tgg aat acc tcc tat 672 Thr Tyr Gln Tyr Lys Ile AlaLeu Asp His Ser Trp Asn Thr Ser Tyr 210 215 220 cca ggt aac aat gta agtctt act gtt cct cag gga ggg gaa aag gtt 720 Pro Gly Asn Asn Val Ser LeuThr Val Pro Gln Gly Gly Glu Lys Val 225 230 235 240 acc ttt acc tat attcca tct acc aac cag gta ttc gat agc gtc aat 768 Thr Phe Thr Tyr Ile ProSer Thr Asn Gln Val Phe Asp Ser Val Asn 245 250 255 cat cct aac caa gcattc cct aca tcc tca gca ggg gtc cag aca aat 816 His Pro Asn Gln Ala PhePro Thr Ser Ser Ala Gly Val Gln Thr Asn 260 265 270 tta gtc caa ttg acttta gcg agt gca ccg gat gtc acc cat aat tta 864 Leu Val Gln Leu Thr LeuAla Ser Ala Pro Asp Val Thr His Asn Leu 275 280 285 gat gta gca gca gacggt tac aaa gcg cac aat att tta cca agg aat 912 Asp Val Ala Ala Asp GlyTyr Lys Ala His Asn Ile Leu Pro Arg Asn 290 295 300 gtt tta aat ctg ccgcgg tat gat tat agt gga aat gat ttg ggt aat 960 Val Leu Asn Leu Pro ArgTyr Asp Tyr Ser Gly Asn Asp Leu Gly Asn 305 310 315 320 gtt tat tca aaggat gca aca tcc ttc cgg gta tgg gct cca aca gct 1008 Val Tyr Ser Lys AspAla Thr Ser Phe Arg Val Trp Ala Pro Thr Ala 325 330 335 tcg aat gtc cagttg ctt tta tac aat agt gag aaa ggt tca ata act 1056 Ser Asn Val Gln LeuLeu Leu Tyr Asn Ser Glu Lys Gly Ser Ile Thr 340 345 350 aaa cag ctt gaaatg caa aag agt gat aac ggt aca tgg aaa ctt cag 1104 Lys Gln Leu Glu MetGln Lys Ser Asp Asn Gly Thr Trp Lys Leu Gln 355 360 365 gtt tct ggt aatctt gaa aac tgg tat tat cta tat caa gtc aca gtg 1152 Val Ser Gly Asn LeuGlu Asn Trp Tyr Tyr Leu Tyr Gln Val Thr Val 370 375 380 aat ggg aca acacaa acg gca gtt gat cca tat gcg cgt gct att tct 1200 Asn Gly Thr Thr GlnThr Ala Val Asp Pro Tyr Ala Arg Ala Ile Ser 385 390 395 400 gtc aat gcaaca cgc ggt atg att gtg gac cta aaa gct acc gat cct 1248 Val Asn Ala ThrArg Gly Met Ile Val Asp Leu Lys Ala Thr Asp Pro 405 410 415 gca ggg tggcag gga gat cat gaa cag aca cct gcg aat cca gta gat 1296 Ala Gly Trp GlnGly Asp His Glu Gln Thr Pro Ala Asn Pro Val Asp 420 425 430 gaa gtg atttat gaa gcg cat gta cgc gat ttt tcg att gat gct aat 1344 Glu Val Ile TyrGlu Ala His Val Arg Asp Phe Ser Ile Asp Ala Asn 435 440 445 tca ggt atgaaa aat aaa ggg aag tat tta gcg ttt aca gag cat gga 1392 Ser Gly Met LysAsn Lys Gly Lys Tyr Leu Ala Phe Thr Glu His Gly 450 455 460 aca aaa ggaccg gat cat gta aag aca ggt att gat agt ttg aag gaa 1440 Thr Lys Gly ProAsp His Val Lys Thr Gly Ile Asp Ser Leu Lys Glu 465 470 475 480 ttg ggcatc acc act gtt caa ttg caa cct gtt gag gag ttt aac agt 1488 Leu Gly IleThr Thr Val Gln Leu Gln Pro Val Glu Glu Phe Asn Ser 485 490 495 att gatgag acc cag cct gat acg tat aac tgg ggc tac gat cca agg 1536 Ile Asp GluThr Gln Pro Asp Thr Tyr Asn Trp Gly Tyr Asp Pro Arg 500 505 510 aac tataac gta cca gag gga gct tat gcc aca act cca gaa gga aca 1584 Asn Tyr AsnVal Pro Glu Gly Ala Tyr Ala Thr Thr Pro Glu Gly Thr 515 520 525 gcg cgtata aca gaa tta aag caa tta att caa agc ctt cat cag cag 1632 Ala Arg IleThr Glu Leu Lys Gln Leu Ile Gln Ser Leu His Gln Gln 530 535 540 cgg attggt gtc aat atg gat gtt gtt tat aac cat acc ttt gat gtg 1680 Arg Ile GlyVal Asn Met Asp Val Val Tyr Asn His Thr Phe Asp Val 545 550 555 560 atggtt tct gat ttt gat aaa att gtc ccg caa tat tat tat cgt acc 1728 Met ValSer Asp Phe Asp Lys Ile Val Pro Gln Tyr Tyr Tyr Arg Thr 565 570 575 gatagt aat ggc aat tat acg aac gga tca ggt tgc ggc aat gaa ttc 1776 Asp SerAsn Gly Asn Tyr Thr Asn Gly Ser Gly Cys Gly Asn Glu Phe 580 585 590 gcgact gag cat cca atg gca caa aag ttt gtg ctt gat tca gtt aat 1824 Ala ThrGlu His Pro Met Ala Gln Lys Phe Val Leu Asp Ser Val Asn 595 600 605 tattgg gta aat gag tac cac gtg gat ggc ttc cgt ttt gac tta atg 1872 Tyr TrpVal Asn Glu Tyr His Val Asp Gly Phe Arg Phe Asp Leu Met 610 615 620 gctctt tta gga aaa gac acg atg gca aaa ata tca aac gag ctg cat 1920 Ala LeuLeu Gly Lys Asp Thr Met Ala Lys Ile Ser Asn Glu Leu His 625 630 635 640gcc att aat cct ggt att gtt tta tat gga gaa cca tgg act ggc ggc 1968 AlaIle Asn Pro Gly Ile Val Leu Tyr Gly Glu Pro Trp Thr Gly Gly 645 650 655aca tcg gga tta tct agc gac cag ctt gta acg aag ggt caa caa aag 2016 ThrSer Gly Leu Ser Ser Asp Gln Leu Val Thr Lys Gly Gln Gln Lys 660 665 670gga tta gga att ggc gtt ttc aac gat aat ata cgt aat ggg ctc gat 2064 GlyLeu Gly Ile Gly Val Phe Asn Asp Asn Ile Arg Asn Gly Leu Asp 675 680 685ggg aac gtg ttt gat aaa acg gca caa ggc ttt gca aca gga gat cca 2112 GlyAsn Val Phe Asp Lys Thr Ala Gln Gly Phe Ala Thr Gly Asp Pro 690 695 700aac cag gtg gat gtc att aaa aat gga gta atc ggt agt att caa gat 2160 AsnGln Val Asp Val Ile Lys Asn Gly Val Ile Gly Ser Ile Gln Asp 705 710 715720 ttt act tca gca cct agc gaa acg att aac tat gtt aca agc cat gat 2208Phe Thr Ser Ala Pro Ser Glu Thr Ile Asn Tyr Val Thr Ser His Asp 725 730735 aac atg acg ctt tgg gat aaa att tta gca agt aat cca agt gac act 2256Asn Met Thr Leu Trp Asp Lys Ile Leu Ala Ser Asn Pro Ser Asp Thr 740 745750 gag gct gac cga att aaa atg gat gaa ttg gca cat gcc gta gta ttc 2304Glu Ala Asp Arg Ile Lys Met Asp Glu Leu Ala His Ala Val Val Phe 755 760765 act tca caa ggt gta cca ttt atg caa ggt gga gaa gaa atg ctg agg 2352Thr Ser Gln Gly Val Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg 770 775780 aca aaa ggc gga aat gat aac agt tat aac gct gga gat agt gtg aat 2400Thr Lys Gly Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Ser Val Asn 785 790795 800 cag ttc gac tgg tca aga aag gcg caa ttt aag gat gtt ttt gac tac2448 Gln Phe Asp Trp Ser Arg Lys Ala Gln Phe Lys Asp Val Phe Asp Tyr 805810 815 ttt tct agt atg att cat ctt cgt aat cag cac ccg gca ttc agg atg2496 Phe Ser Ser Met Ile His Leu Arg Asn Gln His Pro Ala Phe Arg Met 820825 830 acg aca gcg gat caa att aaa cag aat ctt aca ttc tta gaa agc cca2544 Thr Thr Ala Asp Gln Ile Lys Gln Asn Leu Thr Phe Leu Glu Ser Pro 835840 845 aca aac acg gta gct ttc gag tta aag aat tat gca aac cat gat aca2592 Thr Asn Thr Val Ala Phe Glu Leu Lys Asn Tyr Ala Asn His Asp Thr 850855 860 tgg aaa aat ata att gtc atg tat aac cca aat aag act tcc caa acc2640 Trp Lys Asn Ile Ile Val Met Tyr Asn Pro Asn Lys Thr Ser Gln Thr 865870 875 880 ctt aat cta cca agt gga gat tgg acc att gta gga ttg gga gatcaa 2688 Leu Asn Leu Pro Ser Gly Asp Trp Thr Ile Val Gly Leu Gly Asp Gln885 890 895 ata ggt gag aaa tca tta ggg cat gta atg ggt aat gtt caa gtaccg 2736 Ile Gly Glu Lys Ser Leu Gly His Val Met Gly Asn Val Gln Val Pro900 905 910 gct ata agt acg ctt att ctc aaa caa taa 2766 Ala Ile Ser ThrLeu Ile Leu Lys Gln 915 920 2 921 PRT Bacillus acidopullulyticus 2 AspSer Thr Ser Thr Lys Val Ile Val His Tyr His Arg Phe Asp Ser 1 5 10 15Asn Tyr Thr Asn Trp Asp Val Trp Met Trp Pro Tyr Gln Pro Val Asn 20 25 30Gly Asn Gly Ala Ala Tyr Gln Phe Thr Gly Thr Asn Asp Asp Phe Gly 35 40 45Ala Val Ala Asp Thr Gln Val Pro Gly Asp Asn Thr Gln Val Gly Leu 50 55 60Ile Val Arg Lys Asn Asp Trp Ser Glu Lys Asn Thr Pro Asn Asp Leu 65 70 7580 His Ile Asp Leu Ala Lys Gly His Glu Val Trp Ile Val Gln Gly Asp 85 9095 Pro Thr Ile Tyr Tyr Asn Leu Ser Asp Ala Gln Ala Ala Ala Ile Pro 100105 110 Ser Val Ser Asn Ala Tyr Leu Asp Asp Glu Lys Thr Val Leu Ala Lys115 120 125 Leu Ser Met Pro Met Thr Leu Ala Asp Ala Ala Ser Gly Phe ThrVal 130 135 140 Ile Asp Lys Thr Thr Gly Glu Lys Ile Pro Val Thr Ser AlaVal Ser 145 150 155 160 Ala Asn Pro Val Thr Ala Val Leu Val Gly Asp LeuGln Gln Ala Leu 165 170 175 Gly Ala Ala Asn Asn Trp Ser Pro Asp Asp AspHis Thr Leu Leu Lys 180 185 190 Lys Ile Asn Pro Asn Leu Tyr Gln Leu SerGly Thr Leu Pro Ala Gly 195 200 205 Thr Tyr Gln Tyr Lys Ile Ala Leu AspHis Ser Trp Asn Thr Ser Tyr 210 215 220 Pro Gly Asn Asn Val Ser Leu ThrVal Pro Gln Gly Gly Glu Lys Val 225 230 235 240 Thr Phe Thr Tyr Ile ProSer Thr Asn Gln Val Phe Asp Ser Val Asn 245 250 255 His Pro Asn Gln AlaPhe Pro Thr Ser Ser Ala Gly Val Gln Thr Asn 260 265 270 Leu Val Gln LeuThr Leu Ala Ser Ala Pro Asp Val Thr His Asn Leu 275 280 285 Asp Val AlaAla Asp Gly Tyr Lys Ala His Asn Ile Leu Pro Arg Asn 290 295 300 Val LeuAsn Leu Pro Arg Tyr Asp Tyr Ser Gly Asn Asp Leu Gly Asn 305 310 315 320Val Tyr Ser Lys Asp Ala Thr Ser Phe Arg Val Trp Ala Pro Thr Ala 325 330335 Ser Asn Val Gln Leu Leu Leu Tyr Asn Ser Glu Lys Gly Ser Ile Thr 340345 350 Lys Gln Leu Glu Met Gln Lys Ser Asp Asn Gly Thr Trp Lys Leu Gln355 360 365 Val Ser Gly Asn Leu Glu Asn Trp Tyr Tyr Leu Tyr Gln Val ThrVal 370 375 380 Asn Gly Thr Thr Gln Thr Ala Val Asp Pro Tyr Ala Arg AlaIle Ser 385 390 395 400 Val Asn Ala Thr Arg Gly Met Ile Val Asp Leu LysAla Thr Asp Pro 405 410 415 Ala Gly Trp Gln Gly Asp His Glu Gln Thr ProAla Asn Pro Val Asp 420 425 430 Glu Val Ile Tyr Glu Ala His Val Arg AspPhe Ser Ile Asp Ala Asn 435 440 445 Ser Gly Met Lys Asn Lys Gly Lys TyrLeu Ala Phe Thr Glu His Gly 450 455 460 Thr Lys Gly Pro Asp His Val LysThr Gly Ile Asp Ser Leu Lys Glu 465 470 475 480 Leu Gly Ile Thr Thr ValGln Leu Gln Pro Val Glu Glu Phe Asn Ser 485 490 495 Ile Asp Glu Thr GlnPro Asp Thr Tyr Asn Trp Gly Tyr Asp Pro Arg 500 505 510 Asn Tyr Asn ValPro Glu Gly Ala Tyr Ala Thr Thr Pro Glu Gly Thr 515 520 525 Ala Arg IleThr Glu Leu Lys Gln Leu Ile Gln Ser Leu His Gln Gln 530 535 540 Arg IleGly Val Asn Met Asp Val Val Tyr Asn His Thr Phe Asp Val 545 550 555 560Met Val Ser Asp Phe Asp Lys Ile Val Pro Gln Tyr Tyr Tyr Arg Thr 565 570575 Asp Ser Asn Gly Asn Tyr Thr Asn Gly Ser Gly Cys Gly Asn Glu Phe 580585 590 Ala Thr Glu His Pro Met Ala Gln Lys Phe Val Leu Asp Ser Val Asn595 600 605 Tyr Trp Val Asn Glu Tyr His Val Asp Gly Phe Arg Phe Asp LeuMet 610 615 620 Ala Leu Leu Gly Lys Asp Thr Met Ala Lys Ile Ser Asn GluLeu His 625 630 635 640 Ala Ile Asn Pro Gly Ile Val Leu Tyr Gly Glu ProTrp Thr Gly Gly 645 650 655 Thr Ser Gly Leu Ser Ser Asp Gln Leu Val ThrLys Gly Gln Gln Lys 660 665 670 Gly Leu Gly Ile Gly Val Phe Asn Asp AsnIle Arg Asn Gly Leu Asp 675 680 685 Gly Asn Val Phe Asp Lys Thr Ala GlnGly Phe Ala Thr Gly Asp Pro 690 695 700 Asn Gln Val Asp Val Ile Lys AsnGly Val Ile Gly Ser Ile Gln Asp 705 710 715 720 Phe Thr Ser Ala Pro SerGlu Thr Ile Asn Tyr Val Thr Ser His Asp 725 730 735 Asn Met Thr Leu TrpAsp Lys Ile Leu Ala Ser Asn Pro Ser Asp Thr 740 745 750 Glu Ala Asp ArgIle Lys Met Asp Glu Leu Ala His Ala Val Val Phe 755 760 765 Thr Ser GlnGly Val Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg 770 775 780 Thr LysGly Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Ser Val Asn 785 790 795 800Gln Phe Asp Trp Ser Arg Lys Ala Gln Phe Lys Asp Val Phe Asp Tyr 805 810815 Phe Ser Ser Met Ile His Leu Arg Asn Gln His Pro Ala Phe Arg Met 820825 830 Thr Thr Ala Asp Gln Ile Lys Gln Asn Leu Thr Phe Leu Glu Ser Pro835 840 845 Thr Asn Thr Val Ala Phe Glu Leu Lys Asn Tyr Ala Asn His AspThr 850 855 860 Trp Lys Asn Ile Ile Val Met Tyr Asn Pro Asn Lys Thr SerGln Thr 865 870 875 880 Leu Asn Leu Pro Ser Gly Asp Trp Thr Ile Val GlyLeu Gly Asp Gln 885 890 895 Ile Gly Glu Lys Ser Leu Gly His Val Met GlyAsn Val Gln Val Pro 900 905 910 Ala Ile Ser Thr Leu Ile Leu Lys Gln 915920 3 2787 DNA Bacillus deramificans CDS (1)..(2784) 3 gat ggg aac acgaca acg atc att gtc cac tat ttt cgc cct gct ggt 48 Asp Gly Asn Thr ThrThr Ile Ile Val His Tyr Phe Arg Pro Ala Gly 1 5 10 15 gat tat caa ccttgg agt cta tgg atg tgg cca aaa gac gga ggt ggg 96 Asp Tyr Gln Pro TrpSer Leu Trp Met Trp Pro Lys Asp Gly Gly Gly 20 25 30 gct gaa tac gat ttcaat caa ccg gct gac tct ttt gga gct gtt gca 144 Ala Glu Tyr Asp Phe AsnGln Pro Ala Asp Ser Phe Gly Ala Val Ala 35 40 45 agt gct gat att cca ggaaac cca agt cag gta gga att atc gtt cgc 192 Ser Ala Asp Ile Pro Gly AsnPro Ser Gln Val Gly Ile Ile Val Arg 50 55 60 act caa gat tgg acc aaa gatgtg agc gct gac cgc tac ata gat tta 240 Thr Gln Asp Trp Thr Lys Asp ValSer Ala Asp Arg Tyr Ile Asp Leu 65 70 75 80 agc aaa gga aat gag gtg tggctt gta gaa gga aac agc caa att ttt 288 Ser Lys Gly Asn Glu Val Trp LeuVal Glu Gly Asn Ser Gln Ile Phe 85 90 95 tat aat gaa aaa gat gct gag gatgca gct aaa ccc gct gta agc aac 336 Tyr Asn Glu Lys Asp Ala Glu Asp AlaAla Lys Pro Ala Val Ser Asn 100 105 110 gct tat tta gat gct tca aac caggtg ctg gtt aaa ctt agc cag ccg 384 Ala Tyr Leu Asp Ala Ser Asn Gln ValLeu Val Lys Leu Ser Gln Pro 115 120 125 tta act ctt ggg gaa ggc gca agcggc ttt acg gtt cat gac gac aca 432 Leu Thr Leu Gly Glu Gly Ala Ser GlyPhe Thr Val His Asp Asp Thr 130 135 140 gca aat aag gat att cca gtg acatct gtg aag gat gca agt ctt ggt 480 Ala Asn Lys Asp Ile Pro Val Thr SerVal Lys Asp Ala Ser Leu Gly 145 150 155 160 caa gat gta acc gct gtt ttggca ggt acc ttc caa cat att ttt gga 528 Gln Asp Val Thr Ala Val Leu AlaGly Thr Phe Gln His Ile Phe Gly 165 170 175 ggt tcc gat tgg gca cct gataat cac agt act tta tta aaa aag gtg 576 Gly Ser Asp Trp Ala Pro Asp AsnHis Ser Thr Leu Leu Lys Lys Val 180 185 190 act aac aat ctc tat caa ttctca gga gat ctt cct gaa gga aac tac 624 Thr Asn Asn Leu Tyr Gln Phe SerGly Asp Leu Pro Glu Gly Asn Tyr 195 200 205 caa tat aaa gtg gct tta aatgat agc tgg aat aat ccg agt tac cca 672 Gln Tyr Lys Val Ala Leu Asn AspSer Trp Asn Asn Pro Ser Tyr Pro 210 215 220 tct gac aac att aat tta acagtc cct gcc ggc ggt gca cac gtc act 720 Ser Asp Asn Ile Asn Leu Thr ValPro Ala Gly Gly Ala His Val Thr 225 230 235 240 ttt tcg tat att ccg tccact cat gca gtc tat gac aca att aat aat 768 Phe Ser Tyr Ile Pro Ser ThrHis Ala Val Tyr Asp Thr Ile Asn Asn 245 250 255 cct aat gcg gat tta caagta gaa agc ggg gtt aaa acg gat ctc gtg 816 Pro Asn Ala Asp Leu Gln ValGlu Ser Gly Val Lys Thr Asp Leu Val 260 265 270 acg gtt act cta ggg gaagat cca gat gtg agc cat act ctg tcc att 864 Thr Val Thr Leu Gly Glu AspPro Asp Val Ser His Thr Leu Ser Ile 275 280 285 caa aca gat ggc tat caggca aag cag gtg ata cct cgt aat gtg ctt 912 Gln Thr Asp Gly Tyr Gln AlaLys Gln Val Ile Pro Arg Asn Val Leu 290 295 300 aat tca tca cag tac tactat tca gga gat gat ctt ggg aat acc tat 960 Asn Ser Ser Gln Tyr Tyr TyrSer Gly Asp Asp Leu Gly Asn Thr Tyr 305 310 315 320 aca cag aaa gca acaacc ttt aaa gtc tgg gca cca act tct act caa 1008 Thr Gln Lys Ala Thr ThrPhe Lys Val Trp Ala Pro Thr Ser Thr Gln 325 330 335 gta aat gtt ctt ctttat gac agt gca acg ggt tct gta aca aaa atc 1056 Val Asn Val Leu Leu TyrAsp Ser Ala Thr Gly Ser Val Thr Lys Ile 340 345 350 gta cct atg acg gcatcg ggc cat ggt gtg tgg gaa gca acg gtt aat 1104 Val Pro Met Thr Ala SerGly His Gly Val Trp Glu Ala Thr Val Asn 355 360 365 caa aac ctt gaa aattgg tat tac atg tat gag gta aca ggc caa ggc 1152 Gln Asn Leu Glu Asn TrpTyr Tyr Met Tyr Glu Val Thr Gly Gln Gly 370 375 380 tct acc cga acg gctgtt gat cct tat gca act gcg att gca cca aat 1200 Ser Thr Arg Thr Ala ValAsp Pro Tyr Ala Thr Ala Ile Ala Pro Asn 385 390 395 400 gga acg aga ggcatg att gtg gac ctg gct aaa aca gat cct gct ggc 1248 Gly Thr Arg Gly MetIle Val Asp Leu Ala Lys Thr Asp Pro Ala Gly 405 410 415 tgg aac agt gataaa cat att acg cca aag aat ata gaa gat gag gtc 1296 Trp Asn Ser Asp LysHis Ile Thr Pro Lys Asn Ile Glu Asp Glu Val 420 425 430 atc tat gaa atggat gtc cgt gac ttt tcc att gac cct aat tcg ggt 1344 Ile Tyr Glu Met AspVal Arg Asp Phe Ser Ile Asp Pro Asn Ser Gly 435 440 445 atg aaa aat aaaggg aag tat ttg gct ctt aca gaa aaa gga aca aag 1392 Met Lys Asn Lys GlyLys Tyr Leu Ala Leu Thr Glu Lys Gly Thr Lys 450 455 460 ggc cct gac aacgta aag acg ggg ata gat tcc tta aaa caa ctt ggg 1440 Gly Pro Asp Asn ValLys Thr Gly Ile Asp Ser Leu Lys Gln Leu Gly 465 470 475 480 att act catgtt cag ctt atg cct gtt ttc gca tct aac agt gtc gat 1488 Ile Thr His ValGln Leu Met Pro Val Phe Ala Ser Asn Ser Val Asp 485 490 495 gaa act gatcca acc caa gat aat tgg ggt tat gac cct cgc aac tat 1536 Glu Thr Asp ProThr Gln Asp Asn Trp Gly Tyr Asp Pro Arg Asn Tyr 500 505 510 gat gtt cctgaa ggg cag tat gct aca aat gcg aat ggt aat gct cgt 1584 Asp Val Pro GluGly Gln Tyr Ala Thr Asn Ala Asn Gly Asn Ala Arg 515 520 525 ata aaa gagttt aag gaa atg gtt ctt tca ctc cat cgt gaa cac att 1632 Ile Lys Glu PheLys Glu Met Val Leu Ser Leu His Arg Glu His Ile 530 535 540 ggg gtt aacatg gat gtt gtc tat aat cat acc ttt gcc acg caa atc 1680 Gly Val Asn MetAsp Val Val Tyr Asn His Thr Phe Ala Thr Gln Ile 545 550 555 560 tct gacttc gat aaa att gta cca gaa tat tat tac cgt acg gat gat 1728 Ser Asp PheAsp Lys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr Asp Asp 565 570 575 gca ggtaat tat acc aac gga tca ggt act gga aat gaa att gca gcc 1776 Ala Gly AsnTyr Thr Asn Gly Ser Gly Thr Gly Asn Glu Ile Ala Ala 580 585 590 gaa aggcca atg gtt caa aaa ttt att att gat tcc ctt aag tat tgg 1824 Glu Arg ProMet Val Gln Lys Phe Ile Ile Asp Ser Leu Lys Tyr Trp 595 600 605 gtc aatgag tat cat att gac ggc ttc cgt ttt gac tta atg gcg ctg 1872 Val Asn GluTyr His Ile Asp Gly Phe Arg Phe Asp Leu Met Ala Leu 610 615 620 ctt ggaaaa gac acg atg tcc aaa gct gcc tcg gag ctt cat gct att 1920 Leu Gly LysAsp Thr Met Ser Lys Ala Ala Ser Glu Leu His Ala Ile 625 630 635 640 aatcca gga att gca ctt tac ggt gag cca tgg acg ggt gga acc tct 1968 Asn ProGly Ile Ala Leu Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser 645 650 655 gcactg cca gat gat cag ctt ctg aca aaa gga gct caa aaa ggc atg 2016 Ala LeuPro Asp Asp Gln Leu Leu Thr Lys Gly Ala Gln Lys Gly Met 660 665 670 ggagta gcg gtg ttt aat gac aat tta cga aac gcg ttg gac ggc aat 2064 Gly ValAla Val Phe Asn Asp Asn Leu Arg Asn Ala Leu Asp Gly Asn 675 680 685 gtcttt gat tct tcc gct caa ggt ttt gcg aca ggt gca aca ggc tta 2112 Val PheAsp Ser Ser Ala Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu 690 695 700 actgat gca att aag aat ggc gtt gag ggg agt att aat gac ttt acc 2160 Thr AspAla Ile Lys Asn Gly Val Glu Gly Ser Ile Asn Asp Phe Thr 705 710 715 720tct tca cca ggt gag aca att aac tat gtc aca agt cat gat aac tac 2208 SerSer Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Tyr 725 730 735acc ctt tgg gac aaa ata gcc cta agc aat cct aat gat tcc gaa gcg 2256 ThrLeu Trp Asp Lys Ile Ala Leu Ser Asn Pro Asn Asp Ser Glu Ala 740 745 750gat cgg att aaa atg gat gaa ctc gca caa gca gtt gtt atg acc tca 2304 AspArg Ile Lys Met Asp Glu Leu Ala Gln Ala Val Val Met Thr Ser 755 760 765caa ggc gtt cca ttc atg caa ggc ggg gaa gaa atg ctt cgt aca aaa 2352 GlnGly Val Pro Phe Met Gln Gly Gly Glu Glu Met Leu Arg Thr Lys 770 775 780ggc ggc aac gac aat agt tat aat gca ggc gat gcg gtc aat gag ttt 2400 GlyGly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Ala Val Asn Glu Phe 785 790 795800 gat tgg agc agg aaa gct caa tat cca gat gtt ttc aac tat tat agc 2448Asp Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val Phe Asn Tyr Tyr Ser 805 810815 ggg cta atc cac ctt cgt ctt gat cac cca gcc ttc cgc atg acg aca 2496Gly Leu Ile His Leu Arg Leu Asp His Pro Ala Phe Arg Met Thr Thr 820 825830 gct aat gaa atc aat agc cac ctc caa ttc cta aat agt cca gag aac 2544Ala Asn Glu Ile Asn Ser His Leu Gln Phe Leu Asn Ser Pro Glu Asn 835 840845 aca gtg gcc tat gaa tta act gat cat gtt aat aaa gac aaa tgg gga 2592Thr Val Ala Tyr Glu Leu Thr Asp His Val Asn Lys Asp Lys Trp Gly 850 855860 aat atc att gtt gtt tat aac cca aat aaa act gta gca acc atc aat 2640Asn Ile Ile Val Val Tyr Asn Pro Asn Lys Thr Val Ala Thr Ile Asn 865 870875 880 ttg ccg agc ggg aaa tgg gca atc aat gct acg agc ggt aag gta gga2688 Leu Pro Ser Gly Lys Trp Ala Ile Asn Ala Thr Ser Gly Lys Val Gly 885890 895 gaa tcc acc ctt ggt caa gca gag gga agt gtc caa gta cca ggt ata2736 Glu Ser Thr Leu Gly Gln Ala Glu Gly Ser Val Gln Val Pro Gly Ile 900905 910 tct atg atg atc ctt cat caa gag gta agc cca gac cac ggt aaa aag2784 Ser Met Met Ile Leu His Gln Glu Val Ser Pro Asp His Gly Lys Lys 915920 925 taa 2787 4 928 PRT Bacillus deramificans 4 Asp Gly Asn Thr ThrThr Ile Ile Val His Tyr Phe Arg Pro Ala Gly 1 5 10 15 Asp Tyr Gln ProTrp Ser Leu Trp Met Trp Pro Lys Asp Gly Gly Gly 20 25 30 Ala Glu Tyr AspPhe Asn Gln Pro Ala Asp Ser Phe Gly Ala Val Ala 35 40 45 Ser Ala Asp IlePro Gly Asn Pro Ser Gln Val Gly Ile Ile Val Arg 50 55 60 Thr Gln Asp TrpThr Lys Asp Val Ser Ala Asp Arg Tyr Ile Asp Leu 65 70 75 80 Ser Lys GlyAsn Glu Val Trp Leu Val Glu Gly Asn Ser Gln Ile Phe 85 90 95 Tyr Asn GluLys Asp Ala Glu Asp Ala Ala Lys Pro Ala Val Ser Asn 100 105 110 Ala TyrLeu Asp Ala Ser Asn Gln Val Leu Val Lys Leu Ser Gln Pro 115 120 125 LeuThr Leu Gly Glu Gly Ala Ser Gly Phe Thr Val His Asp Asp Thr 130 135 140Ala Asn Lys Asp Ile Pro Val Thr Ser Val Lys Asp Ala Ser Leu Gly 145 150155 160 Gln Asp Val Thr Ala Val Leu Ala Gly Thr Phe Gln His Ile Phe Gly165 170 175 Gly Ser Asp Trp Ala Pro Asp Asn His Ser Thr Leu Leu Lys LysVal 180 185 190 Thr Asn Asn Leu Tyr Gln Phe Ser Gly Asp Leu Pro Glu GlyAsn Tyr 195 200 205 Gln Tyr Lys Val Ala Leu Asn Asp Ser Trp Asn Asn ProSer Tyr Pro 210 215 220 Ser Asp Asn Ile Asn Leu Thr Val Pro Ala Gly GlyAla His Val Thr 225 230 235 240 Phe Ser Tyr Ile Pro Ser Thr His Ala ValTyr Asp Thr Ile Asn Asn 245 250 255 Pro Asn Ala Asp Leu Gln Val Glu SerGly Val Lys Thr Asp Leu Val 260 265 270 Thr Val Thr Leu Gly Glu Asp ProAsp Val Ser His Thr Leu Ser Ile 275 280 285 Gln Thr Asp Gly Tyr Gln AlaLys Gln Val Ile Pro Arg Asn Val Leu 290 295 300 Asn Ser Ser Gln Tyr TyrTyr Ser Gly Asp Asp Leu Gly Asn Thr Tyr 305 310 315 320 Thr Gln Lys AlaThr Thr Phe Lys Val Trp Ala Pro Thr Ser Thr Gln 325 330 335 Val Asn ValLeu Leu Tyr Asp Ser Ala Thr Gly Ser Val Thr Lys Ile 340 345 350 Val ProMet Thr Ala Ser Gly His Gly Val Trp Glu Ala Thr Val Asn 355 360 365 GlnAsn Leu Glu Asn Trp Tyr Tyr Met Tyr Glu Val Thr Gly Gln Gly 370 375 380Ser Thr Arg Thr Ala Val Asp Pro Tyr Ala Thr Ala Ile Ala Pro Asn 385 390395 400 Gly Thr Arg Gly Met Ile Val Asp Leu Ala Lys Thr Asp Pro Ala Gly405 410 415 Trp Asn Ser Asp Lys His Ile Thr Pro Lys Asn Ile Glu Asp GluVal 420 425 430 Ile Tyr Glu Met Asp Val Arg Asp Phe Ser Ile Asp Pro AsnSer Gly 435 440 445 Met Lys Asn Lys Gly Lys Tyr Leu Ala Leu Thr Glu LysGly Thr Lys 450 455 460 Gly Pro Asp Asn Val Lys Thr Gly Ile Asp Ser LeuLys Gln Leu Gly 465 470 475 480 Ile Thr His Val Gln Leu Met Pro Val PheAla Ser Asn Ser Val Asp 485 490 495 Glu Thr Asp Pro Thr Gln Asp Asn TrpGly Tyr Asp Pro Arg Asn Tyr 500 505 510 Asp Val Pro Glu Gly Gln Tyr AlaThr Asn Ala Asn Gly Asn Ala Arg 515 520 525 Ile Lys Glu Phe Lys Glu MetVal Leu Ser Leu His Arg Glu His Ile 530 535 540 Gly Val Asn Met Asp ValVal Tyr Asn His Thr Phe Ala Thr Gln Ile 545 550 555 560 Ser Asp Phe AspLys Ile Val Pro Glu Tyr Tyr Tyr Arg Thr Asp Asp 565 570 575 Ala Gly AsnTyr Thr Asn Gly Ser Gly Thr Gly Asn Glu Ile Ala Ala 580 585 590 Glu ArgPro Met Val Gln Lys Phe Ile Ile Asp Ser Leu Lys Tyr Trp 595 600 605 ValAsn Glu Tyr His Ile Asp Gly Phe Arg Phe Asp Leu Met Ala Leu 610 615 620Leu Gly Lys Asp Thr Met Ser Lys Ala Ala Ser Glu Leu His Ala Ile 625 630635 640 Asn Pro Gly Ile Ala Leu Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser645 650 655 Ala Leu Pro Asp Asp Gln Leu Leu Thr Lys Gly Ala Gln Lys GlyMet 660 665 670 Gly Val Ala Val Phe Asn Asp Asn Leu Arg Asn Ala Leu AspGly Asn 675 680 685 Val Phe Asp Ser Ser Ala Gln Gly Phe Ala Thr Gly AlaThr Gly Leu 690 695 700 Thr Asp Ala Ile Lys Asn Gly Val Glu Gly Ser IleAsn Asp Phe Thr 705 710 715 720 Ser Ser Pro Gly Glu Thr Ile Asn Tyr ValThr Ser His Asp Asn Tyr 725 730 735 Thr Leu Trp Asp Lys Ile Ala Leu SerAsn Pro Asn Asp Ser Glu Ala 740 745 750 Asp Arg Ile Lys Met Asp Glu LeuAla Gln Ala Val Val Met Thr Ser 755 760 765 Gln Gly Val Pro Phe Met GlnGly Gly Glu Glu Met Leu Arg Thr Lys 770 775 780 Gly Gly Asn Asp Asn SerTyr Asn Ala Gly Asp Ala Val Asn Glu Phe 785 790 795 800 Asp Trp Ser ArgLys Ala Gln Tyr Pro Asp Val Phe Asn Tyr Tyr Ser 805 810 815 Gly Leu IleHis Leu Arg Leu Asp His Pro Ala Phe Arg Met Thr Thr 820 825 830 Ala AsnGlu Ile Asn Ser His Leu Gln Phe Leu Asn Ser Pro Glu Asn 835 840 845 ThrVal Ala Tyr Glu Leu Thr Asp His Val Asn Lys Asp Lys Trp Gly 850 855 860Asn Ile Ile Val Val Tyr Asn Pro Asn Lys Thr Val Ala Thr Ile Asn 865 870875 880 Leu Pro Ser Gly Lys Trp Ala Ile Asn Ala Thr Ser Gly Lys Val Gly885 890 895 Glu Ser Thr Leu Gly Gln Ala Glu Gly Ser Val Gln Val Pro GlyIle 900 905 910 Ser Met Met Ile Leu His Gln Glu Val Ser Pro Asp His GlyLys Lys 915 920 925 5 2487 DNA Bacillus acidopullulyticus CDS(1)..(2487) 5 gat tct acc tcg aca gaa gtc att gtg cat tat cat cgt tttgat tct 48 Asp Ser Thr Ser Thr Glu Val Ile Val His Tyr His Arg Phe AspSer 1 5 10 15 aac tat gca aat tgg gat cta tgg atg tgg cca tat caa ccagtt aat 96 Asn Tyr Ala Asn Trp Asp Leu Trp Met Trp Pro Tyr Gln Pro ValAsn 20 25 30 ggt aat gga gca gca tac gag ttt tct gga aag gat gat ttt ggcgtt 144 Gly Asn Gly Ala Ala Tyr Glu Phe Ser Gly Lys Asp Asp Phe Gly Val35 40 45 aaa gca gat gtt caa gtg cct ggg gat gat aca cag gta ggt ctg att192 Lys Ala Asp Val Gln Val Pro Gly Asp Asp Thr Gln Val Gly Leu Ile 5055 60 gtc cgt aca aat gat tgg agc caa aaa aat aca tca gac gat ctc cat240 Val Arg Thr Asn Asp Trp Ser Gln Lys Asn Thr Ser Asp Asp Leu His 6570 75 80 att gat ctg aca aag ggg cat gaa ata tgg att gtt cag ggg gat ccc288 Ile Asp Leu Thr Lys Gly His Glu Ile Trp Ile Val Gln Gly Asp Pro 8590 95 aat att tat tac aat ctg agt gat gcg cag gct gca gcg act cca aag336 Asn Ile Tyr Tyr Asn Leu Ser Asp Ala Gln Ala Ala Ala Thr Pro Lys 100105 110 gtt tcg aat gcg tat ttg gat aat gaa aaa aca gta ttg gca aag cta384 Val Ser Asn Ala Tyr Leu Asp Asn Glu Lys Thr Val Leu Ala Lys Leu 115120 125 act aat cca atg aca tta tca gat gga tca agc ggc ttt acg gtt aca432 Thr Asn Pro Met Thr Leu Ser Asp Gly Ser Ser Gly Phe Thr Val Thr 130135 140 gat aaa aca aca ggg gaa caa att cca gtt acc gct gca aca aat gcg480 Asp Lys Thr Thr Gly Glu Gln Ile Pro Val Thr Ala Ala Thr Asn Ala 145150 155 160 aac tca gcc tcc tcg tct gag cag aca gac ttg gtt caa ttg acgtta 528 Asn Ser Ala Ser Ser Ser Glu Gln Thr Asp Leu Val Gln Leu Thr Leu165 170 175 gcc agt gca ccg gat gtt tcc cat aca ata caa gta gga gca gccggt 576 Ala Ser Ala Pro Asp Val Ser His Thr Ile Gln Val Gly Ala Ala Gly180 185 190 tat gaa gca gtc aat ctc ata cca cga aat gta tta aat ttg cctcgt 624 Tyr Glu Ala Val Asn Leu Ile Pro Arg Asn Val Leu Asn Leu Pro Arg195 200 205 tat tat tac agc gga aat gat tta ggt aac gtt tat tca aat aaggca 672 Tyr Tyr Tyr Ser Gly Asn Asp Leu Gly Asn Val Tyr Ser Asn Lys Ala210 215 220 acg gcc ttc cgt gta tgg gct cca act gct tcg gat gtc caa ttactt 720 Thr Ala Phe Arg Val Trp Ala Pro Thr Ala Ser Asp Val Gln Leu Leu225 230 235 240 tta tac aat agt gaa aca gga cct gta acc aaa cag ctt gaaatg caa 768 Leu Tyr Asn Ser Glu Thr Gly Pro Val Thr Lys Gln Leu Glu MetGln 245 250 255 aag agt gat aac ggt aca tgg aaa ctg aag gtc cct ggt aatctg aaa 816 Lys Ser Asp Asn Gly Thr Trp Lys Leu Lys Val Pro Gly Asn LeuLys 260 265 270 aat tgg tat tat ctc tat cag gta acg gtg aat ggg aag acacaa aca 864 Asn Trp Tyr Tyr Leu Tyr Gln Val Thr Val Asn Gly Lys Thr GlnThr 275 280 285 gcc gtt gac cct tat gtg cgt gct att tca gtc aat gca acacgt ggt 912 Ala Val Asp Pro Tyr Val Arg Ala Ile Ser Val Asn Ala Thr ArgGly 290 295 300 atg ata gtc gat tta gaa gat acg aat cct cct gga tgg aaagaa gat 960 Met Ile Val Asp Leu Glu Asp Thr Asn Pro Pro Gly Trp Lys GluAsp 305 310 315 320 cat caa cag aca cct gcg aac cca gtg gat gaa gta atctac gaa gtg 1008 His Gln Gln Thr Pro Ala Asn Pro Val Asp Glu Val Ile TyrGlu Val 325 330 335 cat gtg cgt gat ttt tcg att gat gct aat tca ggc atgaaa aat aaa 1056 His Val Arg Asp Phe Ser Ile Asp Ala Asn Ser Gly Met LysAsn Lys 340 345 350 ggg aaa tat ctt gcc ttt aca gaa cat ggc aca aaa ggccct gat aac 1104 Gly Lys Tyr Leu Ala Phe Thr Glu His Gly Thr Lys Gly ProAsp Asn 355 360 365 gtg aaa acg ggt att gat agt ttg aag gaa tta gga atcaat gct gtt 1152 Val Lys Thr Gly Ile Asp Ser Leu Lys Glu Leu Gly Ile AsnAla Val 370 375 380 caa tta cag ccg att gaa gaa ttt aac agc att gat gaaacc caa cca 1200 Gln Leu Gln Pro Ile Glu Glu Phe Asn Ser Ile Asp Glu ThrGln Pro 385 390 395 400 aat atg tat aac tgg ggc tat gac cca aga aac tacaac gtc cct gaa 1248 Asn Met Tyr Asn Trp Gly Tyr Asp Pro Arg Asn Tyr AsnVal Pro Glu 405 410 415 gga gcg tat gca act aca cca gaa gga acg gct cgcatt acc cag tta 1296 Gly Ala Tyr Ala Thr Thr Pro Glu Gly Thr Ala Arg IleThr Gln Leu 420 425 430 aag caa ctg att caa agc att cat aaa gat cgg attgct atc aat atg 1344 Lys Gln Leu Ile Gln Ser Ile His Lys Asp Arg Ile AlaIle Asn Met 435 440 445 gat gtg gtc tat aac cat acc ttt aac gta gga gtgtct gat ttt gat 1392 Asp Val Val Tyr Asn His Thr Phe Asn Val Gly Val SerAsp Phe Asp 450 455 460 aag att gtt ccg caa tac tat tat cgg aca gac agcgca ggt aat tat 1440 Lys Ile Val Pro Gln Tyr Tyr Tyr Arg Thr Asp Ser AlaGly Asn Tyr 465 470 475 480 acg aac ggc tca ggt gta ggt aat gaa att gcgacc gag cgt ccg atg 1488 Thr Asn Gly Ser Gly Val Gly Asn Glu Ile Ala ThrGlu Arg Pro Met 485 490 495 gtc caa aag ttc gtt ctg gat tct gtt aaa tattgg gta aag gaa tac 1536 Val Gln Lys Phe Val Leu Asp Ser Val Lys Tyr TrpVal Lys Glu Tyr 500 505 510 cat atc gac ggc ttc cgt ttc gat ctt atg gctctt tta gga aaa gac 1584 His Ile Asp Gly Phe Arg Phe Asp Leu Met Ala LeuLeu Gly Lys Asp 515 520 525 acc atg gcc aaa ata tca aaa gag ctt cat gctatt aat cct ggc att 1632 Thr Met Ala Lys Ile Ser Lys Glu Leu His Ala IleAsn Pro Gly Ile 530 535 540 gtc ctg tat gga gaa cca tgg act ggc ggt acctct gga tta tca agc 1680 Val Leu Tyr Gly Glu Pro Trp Thr Gly Gly Thr SerGly Leu Ser Ser 545 550 555 560 gac caa ctc gtt acg aaa ggt cag caa aagggc ttg gga att ggc gta 1728 Asp Gln Leu Val Thr Lys Gly Gln Gln Lys GlyLeu Gly Ile Gly Val 565 570 575 ttc aac gat aat att cgg aac gga ctc gatggt aac gtt ttt gat aaa 1776 Phe Asn Asp Asn Ile Arg Asn Gly Leu Asp GlyAsn Val Phe Asp Lys 580 585 590 tcg gca caa gga ttt gca aca gga gat ccaaac caa gtt aat gtc att 1824 Ser Ala Gln Gly Phe Ala Thr Gly Asp Pro AsnGln Val Asn Val Ile 595 600 605 aaa aat aga gtt atg gga agt att tca gatttc act tcg gca cct agc 1872 Lys Asn Arg Val Met Gly Ser Ile Ser Asp PheThr Ser Ala Pro Ser 610 615 620 gaa acc att aac tat gta aca agc cat gataat atg aca ttg tgg gat 1920 Glu Thr Ile Asn Tyr Val Thr Ser His Asp AsnMet Thr Leu Trp Asp 625 630 635 640 aaa att agc gca agt aat ccg aac gataca caa gca gat cga att aag 1968 Lys Ile Ser Ala Ser Asn Pro Asn Asp ThrGln Ala Asp Arg Ile Lys 645 650 655 atg gat gaa ttg gct caa gct gtg gtattt act tca caa ggg gta cca 2016 Met Asp Glu Leu Ala Gln Ala Val Val PheThr Ser Gln Gly Val Pro 660 665 670 ttt atg caa ggt gga gaa gaa atg ctgcgg aca aaa ggc ggt aat gat 2064 Phe Met Gln Gly Gly Glu Glu Met Leu ArgThr Lys Gly Gly Asn Asp 675 680 685 aat agt tac aat gcc ggg gat agc gtgaat cag ttc gat tgg tca aga 2112 Asn Ser Tyr Asn Ala Gly Asp Ser Val AsnGln Phe Asp Trp Ser Arg 690 695 700 aaa gca caa ttt gaa aat gta ttc gactac tat tct tgg ttg att cat 2160 Lys Ala Gln Phe Glu Asn Val Phe Asp TyrTyr Ser Trp Leu Ile His 705 710 715 720 cta cgt gat aat cac cca gca ttccgt atg acg aca gcg gat caa atc 2208 Leu Arg Asp Asn His Pro Ala Phe ArgMet Thr Thr Ala Asp Gln Ile 725 730 735 aaa caa aat ctc act ttc ttg gatagc cca acg aac act gta gca ttt 2256 Lys Gln Asn Leu Thr Phe Leu Asp SerPro Thr Asn Thr Val Ala Phe 740 745 750 gaa tta aaa aat cat gcc aat catgat aaa tgg aaa aac att ata gtt 2304 Glu Leu Lys Asn His Ala Asn His AspLys Trp Lys Asn Ile Ile Val 755 760 765 atg tat aat cca aat aaa act gcacaa act ctc act cta cca agt gga 2352 Met Tyr Asn Pro Asn Lys Thr Ala GlnThr Leu Thr Leu Pro Ser Gly 770 775 780 aat tgg aca att gta gga tta ggcaat caa gta ggt gag aaa tca cta 2400 Asn Trp Thr Ile Val Gly Leu Gly AsnGln Val Gly Glu Lys Ser Leu 785 790 795 800 ggc cat gta aat ggc acg gttgag gtg cca gct ctt agt acg atc att 2448 Gly His Val Asn Gly Thr Val GluVal Pro Ala Leu Ser Thr Ile Ile 805 810 815 ctt cat cag ggt aca tct gaagat gtc att gat caa aat 2487 Leu His Gln Gly Thr Ser Glu Asp Val Ile AspGln Asn 820 825 6 829 PRT Bacillus acidopullulyticus 6 Asp Ser Thr SerThr Glu Val Ile Val His Tyr His Arg Phe Asp Ser 1 5 10 15 Asn Tyr AlaAsn Trp Asp Leu Trp Met Trp Pro Tyr Gln Pro Val Asn 20 25 30 Gly Asn GlyAla Ala Tyr Glu Phe Ser Gly Lys Asp Asp Phe Gly Val 35 40 45 Lys Ala AspVal Gln Val Pro Gly Asp Asp Thr Gln Val Gly Leu Ile 50 55 60 Val Arg ThrAsn Asp Trp Ser Gln Lys Asn Thr Ser Asp Asp Leu His 65 70 75 80 Ile AspLeu Thr Lys Gly His Glu Ile Trp Ile Val Gln Gly Asp Pro 85 90 95 Asn IleTyr Tyr Asn Leu Ser Asp Ala Gln Ala Ala Ala Thr Pro Lys 100 105 110 ValSer Asn Ala Tyr Leu Asp Asn Glu Lys Thr Val Leu Ala Lys Leu 115 120 125Thr Asn Pro Met Thr Leu Ser Asp Gly Ser Ser Gly Phe Thr Val Thr 130 135140 Asp Lys Thr Thr Gly Glu Gln Ile Pro Val Thr Ala Ala Thr Asn Ala 145150 155 160 Asn Ser Ala Ser Ser Ser Glu Gln Thr Asp Leu Val Gln Leu ThrLeu 165 170 175 Ala Ser Ala Pro Asp Val Ser His Thr Ile Gln Val Gly AlaAla Gly 180 185 190 Tyr Glu Ala Val Asn Leu Ile Pro Arg Asn Val Leu AsnLeu Pro Arg 195 200 205 Tyr Tyr Tyr Ser Gly Asn Asp Leu Gly Asn Val TyrSer Asn Lys Ala 210 215 220 Thr Ala Phe Arg Val Trp Ala Pro Thr Ala SerAsp Val Gln Leu Leu 225 230 235 240 Leu Tyr Asn Ser Glu Thr Gly Pro ValThr Lys Gln Leu Glu Met Gln 245 250 255 Lys Ser Asp Asn Gly Thr Trp LysLeu Lys Val Pro Gly Asn Leu Lys 260 265 270 Asn Trp Tyr Tyr Leu Tyr GlnVal Thr Val Asn Gly Lys Thr Gln Thr 275 280 285 Ala Val Asp Pro Tyr ValArg Ala Ile Ser Val Asn Ala Thr Arg Gly 290 295 300 Met Ile Val Asp LeuGlu Asp Thr Asn Pro Pro Gly Trp Lys Glu Asp 305 310 315 320 His Gln GlnThr Pro Ala Asn Pro Val Asp Glu Val Ile Tyr Glu Val 325 330 335 His ValArg Asp Phe Ser Ile Asp Ala Asn Ser Gly Met Lys Asn Lys 340 345 350 GlyLys Tyr Leu Ala Phe Thr Glu His Gly Thr Lys Gly Pro Asp Asn 355 360 365Val Lys Thr Gly Ile Asp Ser Leu Lys Glu Leu Gly Ile Asn Ala Val 370 375380 Gln Leu Gln Pro Ile Glu Glu Phe Asn Ser Ile Asp Glu Thr Gln Pro 385390 395 400 Asn Met Tyr Asn Trp Gly Tyr Asp Pro Arg Asn Tyr Asn Val ProGlu 405 410 415 Gly Ala Tyr Ala Thr Thr Pro Glu Gly Thr Ala Arg Ile ThrGln Leu 420 425 430 Lys Gln Leu Ile Gln Ser Ile His Lys Asp Arg Ile AlaIle Asn Met 435 440 445 Asp Val Val Tyr Asn His Thr Phe Asn Val Gly ValSer Asp Phe Asp 450 455 460 Lys Ile Val Pro Gln Tyr Tyr Tyr Arg Thr AspSer Ala Gly Asn Tyr 465 470 475 480 Thr Asn Gly Ser Gly Val Gly Asn GluIle Ala Thr Glu Arg Pro Met 485 490 495 Val Gln Lys Phe Val Leu Asp SerVal Lys Tyr Trp Val Lys Glu Tyr 500 505 510 His Ile Asp Gly Phe Arg PheAsp Leu Met Ala Leu Leu Gly Lys Asp 515 520 525 Thr Met Ala Lys Ile SerLys Glu Leu His Ala Ile Asn Pro Gly Ile 530 535 540 Val Leu Tyr Gly GluPro Trp Thr Gly Gly Thr Ser Gly Leu Ser Ser 545 550 555 560 Asp Gln LeuVal Thr Lys Gly Gln Gln Lys Gly Leu Gly Ile Gly Val 565 570 575 Phe AsnAsp Asn Ile Arg Asn Gly Leu Asp Gly Asn Val Phe Asp Lys 580 585 590 SerAla Gln Gly Phe Ala Thr Gly Asp Pro Asn Gln Val Asn Val Ile 595 600 605Lys Asn Arg Val Met Gly Ser Ile Ser Asp Phe Thr Ser Ala Pro Ser 610 615620 Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Met Thr Leu Trp Asp 625630 635 640 Lys Ile Ser Ala Ser Asn Pro Asn Asp Thr Gln Ala Asp Arg IleLys 645 650 655 Met Asp Glu Leu Ala Gln Ala Val Val Phe Thr Ser Gln GlyVal Pro 660 665 670 Phe Met Gln Gly Gly Glu Glu Met Leu Arg Thr Lys GlyGly Asn Asp 675 680 685 Asn Ser Tyr Asn Ala Gly Asp Ser Val Asn Gln PheAsp Trp Ser Arg 690 695 700 Lys Ala Gln Phe Glu Asn Val Phe Asp Tyr TyrSer Trp Leu Ile His 705 710 715 720 Leu Arg Asp Asn His Pro Ala Phe ArgMet Thr Thr Ala Asp Gln Ile 725 730 735 Lys Gln Asn Leu Thr Phe Leu AspSer Pro Thr Asn Thr Val Ala Phe 740 745 750 Glu Leu Lys Asn His Ala AsnHis Asp Lys Trp Lys Asn Ile Ile Val 755 760 765 Met Tyr Asn Pro Asn LysThr Ala Gln Thr Leu Thr Leu Pro Ser Gly 770 775 780 Asn Trp Thr Ile ValGly Leu Gly Asn Gln Val Gly Glu Lys Ser Leu 785 790 795 800 Gly His ValAsn Gly Thr Val Glu Val Pro Ala Leu Ser Thr Ile Ile 805 810 815 Leu HisGln Gly Thr Ser Glu Asp Val Ile Asp Gln Asn 820 825 7 23 DNA ArtificialSequence Primer 132011 7 cgcttcggaa tcattaggat tgc 23 8 27 DNAArtificial Sequence Primer 132012 8 gcttccgttt tgccttaatg gcgctgc 27 923 DNA Artificial Sequence Primer 136054 9 ggccaaggct ctacccgaac ggc 2310 26 DNA Artificial Sequence Primer 132013 10 gcactttacg gggcgccatggacggg 26 11 43 DNA Artificial Sequence Primer 11 cattctgcag cggccgcaaacgcttattta gatgcttcaa acc 43 12 41 DNA Artificial Sequence Primer 12cattctgcag cggccgcaga tgatcttggg aatacctata c 41 13 35 DNA ArtificialSequence Primer 13 ctttgccacg cagatctctc ccttcgataa aattg 35 14 28 DNAArtificial Sequence Primer 14 cattcaaacg gatccctatc aggcaaag 28 15 27DNA Artificial Sequence Primer 15 gttataatgc acccgatgcg gtcaatg 27 16 32DNA Artificial Sequence Primer 16 cagcaaataa gcccattcca gtgacatctg tg 3217 27 DNA Artificial Sequence Primer 17 cttatttaga tgcatcaccc caggtgc 2718 28 DNA Artificial Sequence Primer 18 caactgcgat cgcaccaagt ggaacgag28 19 27 DNA Artificial Sequence Primer 19 gcgatcgcac cacttggaac gagaggc27 20 29 DNA Artificial Sequence Primer 20 ctgcgatcgc accatttggaacgagaggc 29 21 29 DNA Artificial Sequence Primer 21 gacttttcaattgacccttc ttcgggtat 29 22 32 DNA Artificial Sequence Primer 22gtccgtgact tttcaattga ccctctttcg gg 32 23 36 DNA Artificial SequencePrimer 23 gtccgtgact tttcaattga ccctttttcg ggtatg 36 24 31 DNAArtificial Sequence Primer 24 ccaagatagt tggggttacg atcctcgcaa c 31 2528 DNA Artificial Sequence Primer 25 ccaagatctt tggggttacg atcctcgc 2826 29 DNA Artificial Sequence Primer 26 cccaagattt ttggggttac gatcctcgc29 27 31 DNA Artificial Sequence Primer 27 gtcacaagtc acgatagctacaccctttgg g 31 28 33 DNA Artificial Sequence Primer 28 gtcacaagtcacgatctcta caccctttgg gac 33 29 31 DNA Artificial Sequence Primer 29gtcacaagtc acgatttcta caccctttgg g 31 30 30 DNA Artificial SequencePrimer 30 gcaacgacag tagttataat gccggcgatg 30 31 30 DNA ArtificialSequence Primer 31 gcaacgacct tagttataat gccggcgatg 30 32 30 DNAArtificial Sequence Primer 32 gcaacgactt tagttataat gccggcgatg 30 33 34DNA Artificial Sequence Primer 33 gacttcgata aagcggtacc agaatattat tacc34 34 34 DNA Artificial Sequence Primer 34 gggattacac atgttcatcttatgcctgtt ttcg 34 35 37 DNA Artificial Sequence Primer 35 cattggggtcaacatggatg ttatctataa tcatacc 37 36 35 DNA Artificial Sequence Primer 36gttttcgcat ttaacagtgt cgacgaaact gatcc 35 37 30 DNA Artificial SequencePrimer 37 gacttttcca ttcgcccgaa ttcgggtatg 30 38 33 DNA ArtificialSequence Primer 38 cgtgactttt ccattaaacc gaattcgggt atg 33 39 31 DNAArtificial Sequence Primer 39 ccctagagta acagatgtca ctggaatatc c 31 4032 DNA Artificial Sequence Primer 40 ggatattcca gtgacatctg ttactctagg gg32

1. A method for producing a variant of a parent pullulanase, the varianthaving at least one altered property as compared to the parentpullulanase, the method comprising: a) modeling the parent pullulanaseon the three-dimensional structure of SEQ ID NO: 1 depicted in theAppendix to produce a three-dimensional structure of the parentpullulanase; b) identifying in the three-dimensional structure obtainedin step (a) at least one structural part of the parent pullulanase,wherein an alteration in said structural part is predicted to result inan altered property; c) modifying the nucleic acid sequence encoding theparent pullulanase to produce a nucleic acid sequence encoding adeletion, insertion, or substitution of one or more amino acids at aposition corresponding to said structural part; and d) expressing themodified nucleic acid sequence in a host cell to produce the variantpullulanase.
 2. The method according to claim 1, wherein the alteredproperty is pH dependent activity, thermostability, substrate cleavagepattern, specific activity of cleavage, substrate specificity, such ashigher isoamylase activity and/or substrate binding.
 3. The methodaccording to claim 2, wherein the altered property is a higherisoamylase activity as defined by an increase of at least 5% in thenumber of reducing ends formed in the “assay for isoamylase-likeactivity” described herein, using 50 mM sodium acetate, a pH of 4.5, 5.0or 5.5, a temperature of 60° C. and when incubated with a 10% w/v rabbitliver glycogen solution for a period of 10 min.
 4. The method accordingto claims 1 or 2, wherein the altered property is an improvedthermostability as defined by differential scanning calorimetry (DSC)using the method described herein.
 5. The method according to claims 1or 2, wherein the altered property is an improved thermostability asdefined by an increased half-life (T_(1/2)) of at least about 5%,preferably, at least about 10%, more preferably at least about 15%, morepreferably at least about 25%, most preferably at least about 50%, suchas at least about 100%, in the “T_(1/2) assay for liquefaction”described herein, using a pH of 5.0 and a temperature of 95° C.
 6. Themethod according to claims 1 or 2, wherein the altered property is animproved thermostability as defined by an increased residual enzymeactivity of at least about 5%, preferably, at least about 10%, morepreferably at least about 15%, more preferably at least about 25%, mostpreferably at least about 50%, such as at least about 100%, in the“assay for residual activity after liquefaction” described herein, usinga pH of 5.0 and a temperature of 95° C.
 7. The method according toclaims 1 or 2, wherein the altered property is an improvedthermostability as defined by an increased half-life (T_(1/2)) of atleast about 5%, preferably, at least about 10%, more preferably at leastabout 15%, more preferably at least about 25%, most preferably at leastabout 50%, such as at least about 100%, in the “T_(1/2) assay forsaccharification” described herein, using a pH of 4.5 and a temperatureof 70° C.
 8. The method according to claims 1 or 2, wherein the alteredproperty is an improved thermostability as defined by an increasedresidual enzyme activity of at least about 5%, preferably, at leastabout 10%, more preferably at least about 15%, more preferably at leastabout 25%, most preferably at least about 50%, such as at least about100%, in the “assay for residual activity after saccharification”described herein, using a pH of 4.5 and a temperature of 63° C.
 9. Themethod according to claim 8, wherein the “assay for activity forsaccharification” described herein, is carried out at a pH of 4.5 and ata temperature of 70° C.
 10. A method for constructing a variant of aparent pullulanase, the method comprising: a) identifying an internal orexternal cavity or crevice in the three-dimensional structure of theparent pullulanase; b) substituting at least one amino acid residue inthe neighborhood of the cavity or crevice with another amino acidresidue which increases the hydrophobic interaction and/or fills out orreduces the size of the cavity or crevice; c) optionally repeating stepsa) and b) recursively; d) optionally, making alterations each of whichis an insertion, a deletion or a substitution of an amino acid residueat one or more positions other than b); e) preparing the variantresulting from steps a)-d); f) testing the thermostability of saidvariant; and g) optionally repeating steps a)-f) recursively; and h)selecting a variant having increased thermostability as compared to theparent pullulanase.
 11. A method for constructing a variant of a parentpullulanase, the method comprising: a) identifying in thethree-dimensional structure of the parent pullulanase two or more aminoacid residues which, when substituted with cysteines, are capable offorming a disulfide bond; b) substituting the amino acids identified ina) with cysteines; c) optionally repeating steps a) and b) recursively;d) optionally, making alterations each of which is an insertion, adeletion or a substitution of an amino acid residue at one or morepositions other than b); e) preparing the variant resulting from stepsa)-d); f) testing the thermostability of said variant; and g) optionallyrepeating steps a)-f) recursively; and h) selecting a variant havingincreased thermostability as compared to the parent pullulanase.
 12. Amethod for constructing a variant of a parent pullulanase, the methodcomprising: a) identifying, on the surface of the parent pullulanase, atleast one amino acid residue selected from the group consisting of Asp,Glu, Arg, Lys and His; b) substituting, on the surface of the parentpullulanase, at least one amino acid residue selected from the groupconsisting of Asp, Glu, Arg, Lys and His with an uncharged amino acidresidue. c) optionally repeating steps a) and b) recursively; d)optionally, making alterations each of which is an insertion, a deletionor a substitution of an amino acid residue at one or more positionsother than b); e) preparing the variant resulting from steps a)-d); f)testing the thermostability of said variant; and g) optionally repeatingsteps a)-f) recursively; and h) selecting a variant having increasedthermostability as compared to the parent pullulanase.
 13. A method forconstructing a variant of a parent pullulanase, the method comprising:a) identifying an amino acid sequence which links together two or moredomains of the parent pullulanase together; b) substituting at least oneamino acid residue in the said amino acid sequence with another aminoacid residue or deleting at least one amino acid residue in said aminoacid sequence; c) optionally repeating steps a) and b) recursively; d)optionally, making alterations each of which is an insertion, a deletionor a substitution of an amino acid residue at one or more positionsother than b); e) preparing the variant resulting from steps a)-d); f)testing the thermostability of said variant; and g) optionally repeatingsteps a)-f) recursively; and h) selecting a variant having increasedthermostability as compared to the parent pullulanase.
 14. A methodaccording to claim 13, the method comprising in step b) deleting atleast one amino acid residue in said amino acid sequence;
 15. A methodaccording to any of claims 10-14, wherein the increased thermostabilityis as defined in any of claims 4-9.
 16. A method for constructing avariant of a parent pullulanase, where the variant pullulanse has analtered substrate specificity as compared to the parent pullulanase, themethod comprising: a) identifying the substrate binding area in a modelof the three-dimensional structure of the parent pullulanase; b)modifying the substrate binding area by an amino acid substitution,deletion and/or insertion; c) optionally repeating step b) recursively;d) optionally, making alterations each of which is an insertion, adeletion or a substitution of an amino acid residue at one or morepositions other than b), e) preparing the variant resulting from stepsa)-d); f) testing the substrate specificity of the variant; g)optionally repeating steps a)-f) recursively; and h) selecting a varianthaving an altered substrate specificity as compared to the parentpullulanase.
 17. The method according to claim 16, wherein the alteredsubstrate specificity is an increased isoamylase activity compared tothe parent pullulanase.
 18. The method according to claim 17, whereinthe increased isoamylase activity is defined by an increase of at least5% in the number of reducing ends formed in the “assay forisoamylase-like activity” described herein, using 50 mM sodium acetate,a pH of 4.5, 5.0 or 5.5, a temperature of 60° C. and when incubated witha 10% w/v rabbit liver glycogen solution for a period of 10 min.
 19. Amethod for constructing a variant of a parent pullulanase, the methodcomprising: a) identifying an amino acid residue which is within 15 Å,in particular 10 Å, from an active site residue of the parentpullulanase in the three-dimensional structure of said parentpullulanse, and which is involved in electrostatic or hydrophobicinteractions with an active site residue; b) substituting said aminoacid residue with another amino acid residue which changes theelectrostatic and/or hydrophobic surroundings of an active site residue,and which can be accommodated in the structure; c) optionally repeatingsteps a) and b) recursively; d) optionally, making alterations each ofwhich is an insertion, a deletion or a substitution of an amino acidresidue at one or more positions other than b); e) preparing the variantresulting from steps a)-d); f) testing the pH dependent activity of saidvariant; and g) optionally repeating steps a)-f) recursively; and h)selecting a variant having an altered pH dependent activity as comparedto the parent amylase.
 20. A method according to any of the precedingclaims, wherein the parent pullulanase has more than 40% homology withthe amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ IDNO: 5, preferably more than 50%, such as more than 60%, more than 70%,more than 75%, more than 80%, more than 85%, more than 90%, more than91%, more than 92%, more than 93%, more than 94%, more than 95%, morethan 96%, more than 97%, more than 98%, more than 99% homology with theamino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.21. A method according to claim 20, wherein the parent pullulanase hasthe amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ IDNO:
 5. 22. A method for producing a pullulanase variant, the methodcomprising: a) constructing the variant by the method according to anyof claims 10-21; b) transforming a microorganism with a DNA sequenceencoding the variant; c) cultivating the transformed microorganism underconditions which are conducive for producing the variant; and d)optionally, recovering the variant from the resulting culture broth. 23.A pullulanase variant, wherein (a) the variant has more than 40%homology to SEQ ID NO:1; (b) the variant comprises an amino acidmodification compared to SEQ ID NO:1 in at least one of the positionscorresponding to 95-113, K122P, 130-140, K151P, 157-165, 180, 181, 210,227, 228, 232-238, 259, 266-272, 286, G293P, 298, 299, 300-314, such as302-308, N315P, 337-339, 353, N374P, 380, 384, 385, 392, 394, 396, 406,408-429, such as 418-428, 442, A446P, 478, 500-507, 515, 526, 534, 543,544 550, T556P, 557, 563, 568, 573, 576, 583, 627, 659-665, G668P,G672P, 681, 684, 688, 689, 751-755, 732, 736, 740, 760, 767, 770 783,788, 792, 793, such as N793P, K758C+1914C, T916C+A765C, I897C+S819C,P525C+E499C and H286C+T148C; (c) the variant has an improvedthermostability as compared to the parent pullulanase.
 24. A pullulanasevariant, wherein (a) the variant has more than 40% homology to SEQ IDNO:1; (b) the variant comprises an amino acid modification compared toSEQ ID NO:1 in at least one of the positions corresponding to 437, 439,487, 489, 490, 494-496, 505-511, 514, 551-559, 584-590, 620-626,650-658, 665-668, 679, 681, 684, 685, 690-693, 731, 734-738, 775, 786,and 789-795; (c) the variant has an increased isoamylase activity ascompared to the parent pullulanase.
 25. A pullulanase variant, wherein(a) the variant has more than 40% homology to SEQ ID NO:1; (b) thevariant comprises an amino acid modification compared to SEQ ID NO: 1 inat least one of the positions corresponding to 430, 433, 435-443,486-496, 505-515, 518, 521, 548-560, 565, 573-575, 583-595, 599, 600,602-604, 606-608, 610, 611, 616-633, 635, 636, 639, 646-672, 674-696,717, 720-722, 725-747, 760, 763, 764, 767, 773-781, 783-797, 799-802 and817; © the variant has an altered pH dependent activity as compared tothe parent pullulanase.
 26. A pullulanase variant according to any ofclaims 23, 24 or 25, wherein the variant has more than 45% homology withthe amino acid sequence shown in SEQ ID NO: 1, preferably more than 50%,such as more than 60%, more than 70%, more than 75%, more than 80%, morethan 85%, more than 90%, more than 91%, more than 92%, more than 93%,more than 94%, more than 95%, more than 96%, more than 97%, more than98%, more than 99% homology with the amino acid sequence shown in SEQ IDNO:
 1. 27. A pullulanase variant according to claim 26, wherein theparent pullulanase has the amino acid sequence shown in SEQ ID NO: 1.28. A pullulanase variant, wherein (a) the variant has more than 40%homology to SEQ ID NO:3; (b) the variant comprises an amino acidmodification compared to SEQ ID NO:3 in at least one of the positionscorresponding to 111, 112, 158-160, 270-274, 302-314, 392, 400, 408-426,428, 437, 440, 444, 446, 483, 485, 487, 492, 495, 504, 551, D526P, 530,543, 566, 613, 621, 710, 717, 735, 775, 779, 789, G794P, 823, 855, 891,892, 437+503 and 674+664; (c) the variant has an improvedthermostability as compared to the parent pullulanase.
 29. A pullulanasevariant, wherein (a) the variant has more than 40% homology to SEQ IDNO:3; (b) the variant comprises an amino acid modification compared toSEQ ID NO:3 in at least one of the positions corresponding to 435, 437,485, 487, 488, 492-494, 503-509, 512, 549-557, 582-588, 618-624,648-656, 663-666, 677, 679, 682, 683, 688-691, 729, 732-736, 773, 784,787-793; (c) the variant has an increased isoamylase activity ascompared to the parent pullulanase.
 30. A pullulanase variant, wherein(a) the variant has more than 40% homology to SEQ ID NO:3; (b) thevariant comprises an amino acid modification compared to SEQ ID NO: 3 inat least one of the positions corresponding to 428, 431, 433-441,484-494, 503-513, 516, 519, 546-558, 563, 571-573, 581-593, 597, 598,600-602, 604-606, 608, 609, 614-631, 633, 634, 637, 644-670, 672-694,715, 718-720, 723-745, 758, 761, 762, 765, 771-779, 781-795, 797-800,and 815; (c) the variant has an altered pH dependent activity ascompared to the parent pullulanase.
 31. A pullulanase variant accordingto any of claims 28, 29 or 30, wherein the variant has more than 45%homology with the amino acid sequence shown in SEQ ID NO: 3, preferablymore than 50%, such as more than 60%, more than 70%, more than 75%, morethan 80%, more than 85%, more than 90%, more than 91%, more than 92%,more than 93%, more than 94%, more than 95%, more than 96%, more than97%, more than 98%, more than 99% homology with the amino acid sequenceshown in SEQ ID NO:
 3. 32. A pullulanase variant according to claim 31,wherein the parent pullulanase has the amino acid sequence shown in SEQID NO:
 3. 33. A variant according to claims 23 or 28, wherein theimproved thermostability is defined by an increased half-life (T_(1/2))of at least about 5%, preferably, at least about 10%, more preferably atleast about 15%, more preferably at least about 25%, most preferably atleast about 50%, such as at least about 100%, in the “T_(1/2) assay forliquefaction” described herein, using a pH of 5.0 and a temperature of95° C.
 34. A variant according to claims 23 or 28, wherein the improvedthermostability is defined by an increased residual enzyme activity ofat least about 5%, preferably, at least about 10%, more preferably atleast about 15%, more preferably at least about 25%, most preferably atleast about 50%, such as at least about 100%, in the “assay for residualactivity after liquefaction” described herein, using a pH of 5.0 and atemperature of 95° C.
 35. A variant according to claims 23 or 28,wherein the improved thermostability is defined by an increasedhalf-life (T_(1/2)) of at least about 5%, preferably, at least about10%, more preferably at least about 15%, more preferably at least about25%, most preferably at least about 50%, such as at least about 100%, inthe “T_(1/2) assay for saccharification” described herein, using a pH of4.5 and a temperature of 70° C.
 36. A variant according to claims 23 or28, wherein the improved thermostability is defined by an increasedresidual enzyme activity of at least about 5%, preferably, at leastabout 10%, more preferably at least about 15%, more preferably at leastabout 25%, most preferably at least about 50%, such as at least about100%, in the “assay for residual activity after saccharification”described herein, using a pH of 4.5 and a temperature of 63° C.
 37. Avariant according to claim 36, wherein the “assay for activity forsaccharification” described herein, is carried out at a pH of 4.5 and ata temperature of 70° C.
 38. A variant according to claims 24 or 29,wherein the increased isoamylase activity is defined by an increase ofat least 5% in the number of reducing ends formed in the “assay forisoamylase-like activity” described herein, using 50 mM sodium acetate,a pH of 4.5, 5.0 or 5.5, a temperature of 60° C. and when incubated witha 10% w/v rabbit liver glycogen solution for a period of 10 min.
 39. Avariant according to any of claims 23, 25, 28 or 30, wherein the variantfurther has an increased isoamylase activity as compared to the parentpullulanase.
 40. A variant according to claim 37, wherein the increasedisoamylase activity is as defined in claim
 38. 41. A variant accordingto any of claims 24, 25, 28 or 30, wherein the variant further has animproved thermostability as compared to the parent pullulanase.
 42. Avariant according to claim 41, wherein the improved thermostability isas defined in any of claims 33-37.
 43. A variant according to any ofclaims 23, 24, 28 or 29, wherein the variant further has an altered pHdependent activity as compared to the parent pullulanase.
 44. Anisolated nucleic acid sequence comprising a nucleic acid sequence, whichencodes for the pullulanase variant defined in any of claims 23-43. 45.An isolated nucleic acid sequence according to claim 44, wherein thenucleic acid sequence is selected form the group consisting of: (a) anucleic acid sequence having at least 40% homology with the nucleic acidsequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, and (b) a nucleic acidsequnce which hybridizes under low stringency conditions, preferablyunder medium stringency conditions, in particular under high stringencyconditions, with © a complementary strand of the nucleic acid sequenceshown in SEQ ID NO: 1 or SEQ ID NO: 3, or (d) a subsequence of (i) of atleast 100 nucleotides.
 46. An isolated nucleic acid sequence accordingto claim 45, wherein the nucleic acid sequence has at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% homology with the nucleic acidsequence shown as SEQ ID NO: 1 or SEQ ID NO:3.
 47. An isolated nucleicacid construct comprising a nucleic acid sequence as defined in any ofclaims 44-46, operably linked to one or more control sequences capableof directing the expression of the polypeptide in a suitable expressionhost.
 59. Use of a variant of claims 23 to 43 in a detergentcomposition.